WO2006047614A2 - Compositions et procedes d'analyse de biomolecules au moyen de la spectroscopie de masse - Google Patents
Compositions et procedes d'analyse de biomolecules au moyen de la spectroscopie de masse Download PDFInfo
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- WO2006047614A2 WO2006047614A2 PCT/US2005/038623 US2005038623W WO2006047614A2 WO 2006047614 A2 WO2006047614 A2 WO 2006047614A2 US 2005038623 W US2005038623 W US 2005038623W WO 2006047614 A2 WO2006047614 A2 WO 2006047614A2
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
Definitions
- the present invention relates to the analysis of proteins and other biopolymers using mass spectroscopy (MS), particularly for matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography mass spectrometry (LC/MS).
- MS mass spectroscopy
- MALDI-TOF MS matrix-assisted laser desorption time-of-flight mass spectrometry
- LC/MS liquid chromatography mass spectrometry
- the invention is drawn to mass spectroscopy.
- mass spectrometry encompasses any spectrometric technique or process in which molecules are ionized and separated and/or analyzed based on their respective molecular weights.
- mass spectrometry encompasses any type of ionization method, including without limitation electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI) and other forms of atmospheric pressure ionization (API), and laser irradiation.
- ESI electrospray ionization
- APCI atmospheric-pressure chemical ionization
- API atmospheric pressure ionization
- laser irradiation laser irradiation.
- Mass spectrometers are commonly combined with separation methods such as gas chromatography (GC) and liquid chromatography (LC).
- MS/MS is an analogous technique where the first-stage separation device is another mass spectrometer.
- the separation methods comprise liquid chromatography and MS. Any combination (e.g., GC/MS/MS, GC/LC/MS, GC/LC/MS/MS, etc.) of methods can be used to practice the invention.
- MS can refer to any form of mass spectrtometry; by way of non-limiting example, "LC/MS” encompasses LC/ESI MS and LC/MALDI-TOF MS.
- mass spectrometry and “MS” include without limitation APCI MS; ESI MS; GC MS; MALDI-TOF MS; LC/MS combinations; LC/MS/MS combinations; MS/MS combinations; etc. HPLC and RP-HPLC
- MS MS-chromatography
- High-pressure liquid chromatography is a separative and quantitative analytical tool that is generally robust, reliable and flexible.
- Reverse-phase is a commonly used stationary phase that is characterized by alkyl chains of specific length immobilized to a silica bead support.
- RP-HPLC is suitable for the separation and analysis of various types of compounds including without limitation biomolecules, (e.g., carbohydrates, proteins, peptides, and nucleic acids).
- biomolecules e.g., carbohydrates, proteins, peptides, and nucleic acids.
- ESI electrospray ionization
- liquid samples can be introduced into a mass spectrometer by a process that creates multiple charged ions (WiIm et al., Anal. Chem. 68:1, 1996). However, multiple ions can result in complex spectra and reduced sensitivity.
- aqueous buffer is used to elute the salts, while the peptides and proteins are eluted with a mixture of aqueous solvent (water) and organic solvent (acetonitrile, methanol, propanol).
- the aqueous phase is generally HPLC grade water with 0.1% acid and the organic solvent phase is generally an HPLC grade acetonitrile or methanol with 0.1% acid.
- the acid is used to improve the chromatographic peak shape and to provide a source of protons in reverse phase LC/MS.
- the acids most commonly used are formic acid, triflouroacetic acid, and acetic acid.
- RP HPLC compounds are separated based on their hydrophobic character.
- MALDI-TOF MS matrix-assisted laser desorption time-of- flight mass spectrometry
- MALDI-TOF MS matrix-assisted laser desorption time-of- flight mass spectrometry
- Matrix molecules transfer their energy to analyte molecules, causing them to vaporize and ionize.
- the ionized molecules are accelerated in an electric field and enter the flight tube. During the flight in this tube, different molecules are separated according to their mass to charge (m/z) ratio and reach the detector at different times. Each molecule yields a distinct signal.
- the method is used for detection and characterization of biomolecules, such as proteins, peptides, oligosaccharides and oligonucleotides, with molecular masses between about 400 and about 500,000 Da, or higher.
- MALDI-MS is a sensitive technique that allows the detection of low (10-15 to 10-18 mole) quantities of analyte in a sample.
- Partial amino acid sequences of proteins can be determined by enzymatic proteolysis followed by MS analysis of the product peptides. These amino acid sequences can be used for in silico examination of DNA and/or protein sequence databases. Matched amino acid sequences can indicate proteins, domains and/or motifs having a known function and/or tertiary structure. For example, amino acid sequences from an uncharacterized protein might match the sequence or structure of a domain or motif that binds a ligand. As another example, the amino acid sequences can be used in vitro as antigens to generate antibodies to the protein and other related proteins from other biological source material (e.g., from a different tissue or organ, or from another species).
- MS MS
- MALDI-TOF MS MS-TOF MS
- MALDI-TOF MS is a powerful technique, it has its limitations. Non- limiting examples of such limitations involve adduction, solubilization, and a limited analyzable surface area. These and other factors increase the amount of "noise" in MS spectra.
- MALDI-MS One limitation to MALDI-MS is the process of adduction, in which ions form adducts that interfere with MALDI-TOF mass spectroscopy.
- ions form adducts that interfere with MALDI-TOF mass spectroscopy.
- sodium, potassium, ammonium and other monovalent cations are known to cause adducts that interfere with MALDI-TOF mass spectroscopy, generally when present in a range of from about 10 to about 750 mM, more specifically from about 50 to about 500 mM.
- Protein adducts are particularly undesirable to those studying a proteome: the transformation and loss of molecules of interest results in the production of adducts, which are undesirable contaminants. Both events complicate the target sample, and both can introduce inaccuracy and/or imprecision in the MS spectra.
- sample complexity can result in less sensitive and accurate results.
- Sample complexity reflects a number of factors but it generally increases as the number of different molecular species in a sample increases and as the concentration of undesirable molecular species (i.e., molecules other than the molecule of interest) increases.
- One source of sample complexity is adduction of monovalent cations to peptides. This leads to the formation of peptide:ion adducts with ions.
- monovalent cations such as sodium and potassium ions are undesirable contaminants that originate from commonly used buffers or from incompletely deionized water.
- Adducts reduce the sensitivity of the analysis by partitioning the signal intensity arising from a single peptide into various adduct cluster peaks. This phenomenon is especially problematic for enzymatic digests of low abundance proteins. Adduct clusters can also suppress the signal of an overlapping or neighboring peak of low abundance. In the extreme this could result in lower confidence for protein identification resulting in missed protein identifications and/or lower sequence coverage.
- PTM post-translational modification
- peptides co-spotted and co-crystallized with MALDI matrix can be washed on the MALDI target using solvent or water to remove excess salts (Vorm et al., Anal. Chem. 66:3281, 1994), but this protocol can also result in significant loss of low abundance peptides.
- samples can be desalted by reverse-phase separation via an HPLC where the eluted fractions are spotted (either manually or by a robotic device) onto MALDI target surfaces.
- Another method is to displace monovalent metal cations with a volatile monovalent cation such as ammonium (Cheng et al., Rapid Commun Mass Spectrom 10:907, 1996), however this involves introduction of yet another salt into the sample, which may lead to overall signal suppression and extensive formation of matrix clusters.
- a volatile monovalent cation such as ammonium
- solubilizing agents useful in the analysis of hydrophobic analytes, interfere with MALDI-TOF MS and other types of mass spectroscopy.
- the analytes are proteins, some of which are readily soluble in water, some of which are not.
- MS-compatible generally indicates a composition that can be used in MALDI-TOF MS experiments. More specifically, a MS-compatible solubilizer preferably does not (1) interfere with the co- precipitation of analyte and matrix molecules, (2) impede the transfer of energy from matrix to analyte molecules, (3) lower the ionization efficiency of analyte molecules, and/or (4) increase the number of ion adducts.
- MS-compatible compositons and techniques for purifying analytes for MS preferably have the further desirable characteristic of (5) not adhering or causing damage to the column and/or instrument tubing during and after appropriate washing procedures.
- MS-compatible solubilizers and other compositions such as blends of detergents and/or non-detergent surfactants and blends (mixtures) thereof, that may be used to sequester monovalent cation adducts are disclosed herein.
- Application of the MS-compatible solubilizers of the invention as a matrix additive for MALDI-TOF-MS analysis reduces the complexity of sodium-rich peptide samples without affecting the sensitivity of the analysis.
- the signal-to-noise ratio (a.k.a. SNR) is the ratio of the intensity of a signal (meaningful information) to the intensity of background "noise". Data with higher SNR are desirable as they are "cleaner", i.e., they have a higher density of information.
- buffer salt additives such as ammonium citrate or ammonium phosphate
- MALDI-MS standards from Applied Biosystems recommend mixing into a matrix solution containing 50 mM ammonium phosphate or ammonium citrate.
- MALDI-MS analysis of oligonucleotides can be carried using buffer additives such as tetraamine spermine (Asara et al., Anal. Chem. 71:2866, 1999), fucose (Distler et al., Anal. Chem.
- Another method of removing salt contaminants is to wash the spotted anal yte: matrix mix with cold water.
- this method can lead to losses of small polar peptides, or peptides modified with polar moieties.
- it is generally not useful for quantitative studies intended to measure the efficiency of a post-translational modification, as one or more of the forms may be washed out.
- the invention provides compositions and methods useful in mass spectrometry (MS), including without limitation LC/MS and MALDI-TOF MS, often referred to herein simply as MALDI MS.
- the invention provides reagents for use in preparing target molecules for mass spectrometry, in which the reagents are mass spectrometry compatible ("MS-compatible"), meaning that they do not reduce the quality of the mass spectra obtained when a target molecule is analyzed by MS.
- MS-compatible mass spectrometry compatible
- the invention further, provides reagents that improve the quality of mass spectra obtained by MS analysis, such as but not limited to MALDI MS.
- MS-compatible solubilizers that can increase the solubility of an analyte.
- the MS-compatible solubilizers can include without limitation detergents or surfactants.
- Blends of solubilizers are included, in which the blends can include one or more detergents or one or more surfactants, and one one or more detergents in combination with one or more surfactants.
- the solubilizer blends can optionally further include buffers, chaotropic agents, salts, or other chemical entities.
- the solubilizers and solubilizer blends can be present during mass spectrometry analysis, such as by MALDI MS or LC/MS.
- the invention provides MS-compatible sorbents.
- the invention provides MALDI matrix additives that support and/or promote the formation of small analyte:matrix crystals in thin layers.
- MS-compatible sorbents are silicas, aluminia, germanium oxide, indium tin oxide, metal oxides, chlorides, sulfates, phosphates, carbonates and fluorides; polymer based oxides, chlorides, sulfates, carbonates, phosphates or fluorides; diatomaceous earth; graphite or activated charcoal; and titania, gold and activated gold.
- the invention provides MS-compatible buffers.
- the MS-compatible buffer is a morpholino-sulfonic acid, such as 2-(n- morpholino)ethane sulfonic acid (MES); 4-(n-morpholino)butane-sulfonic acid (MOBS); 3- (n-morpholino)propane-sulfonic acid (MOPS); or 3-(n-morpholino)-2- hydroxypropanesulfonic acid (MOPSO).
- MES 2-(n- morpholino)ethane sulfonic acid
- MOBS 4-(n-morpholino)butane-sulfonic acid
- MOPS 3- (n-morpholino)propane-sulfonic acid
- MOPSO 3-(n-morpholino)-2- hydroxypropanesulfonic acid
- the invention also provides compositions and methods of preparing a hydrophobic molecule for MALDI-TOF MS analysis, the methods comprising contacting a composition comprising said hydrophobic molecule with at least one MS-compatible solubilizer.
- the invention also provides compositions and methods for performing LC/MS analysis of a sample, the methods comprising contacting a sample with at least one MS- compatible solubilizer and performing LC/MS analysis of a sample.
- the invention provides methods of performing isoelectric focusing of a sample, where the sample has been contacted with at least one MS-compatible solubilizer. The sample or a portion thereof, can be analyzed by mass spectrometry after isoelectric focusing has been performed.
- the invention also provides compositions and methods for preparing a protein for MALDI-TOF analysis, the method comprising contacting a composition comprising the protein, in any order or combination, with (a) at least one MALDI matrix additive of the invention and (b) at least one enzyme, such as a protease or a protein-modifying enzyme.
- a protease or a protein-modifying enzyme.
- the enzyme is a protease.
- the invention provides compositions and methods for preparing a protein having one or more hydrophobic regions for MALDI-TOF analysis, the method comprising contacting a composition comprising said protein, in any order or combination, with (a) at least one MS-compatible solubilizer and (b) at least one enzyme, such as a protease or a protein-modifying enzyme.
- the invention provides compositions and methods of preparing a sample comprising a protein, such methods comprising (a) subjecting a sample comprising the protein to a process that at least partially separates the protein from other molecules in the sample, to generate a partially purified protein, and (b) contacting a composition comprising the partially purified protein with MALDI matrix additive of the invention, thereby generating a sample comprising the protein suitable for MALDI-TOF analysis.
- An enzyme such as a protease, and/or a matrix suitable for MALDI-TOF may also be added at (b) or at some other point in sample preparation.
- a preferred MALDI matrix additive is a MS-compatible solubilizer.
- the invention provides compositions and methods for identifying a region on a molecule that binds to a region on a ligand, comprising contacting the molecule and/or the ligand with at least one MALDI matrix additive of the invention, thereby generating a sample suitable for MALDI-TOF analysis, and subjecting the sample to MALDI-TOF analysis.
- the molecule and/or the ligand can be hydrophobic, or comprise at least one region that is hydrophobic, in which case a preferred MALDI matrix additive is a MS-compatible solubilizer of the invention.
- the invention provides compositions and methods for identifying a protein that binds to a ligand, the method comprising (a) contacting, in any order or combination, (i) a sample comprising one or more proteins, (ii) the ligand, (iii) one or more cross-linkers, (iv) a MALDI matrix additive of the invention, and (v) a protease; in order to generate cross-linked peptides, which are cross-linked to the ligand or some portion thereof, and determining the amino acid sequences of the cross-linked peptides by MALDI- MS analysis.
- the amino acid sequence of the cross-linked peptides comprise all or part of a region on a protein that binds to said ligand.
- the invention provides compositions and methods for identifying a protein or region thereof that is chemically modified, said method comprising: (a) contacting, in any order or combination, (i) the protein, (ii) an enzyme that modifies proteins, (iii) a MALDI matrix additive of the invention, and (iv) a protease, in order to generate chemically modified peptides.
- the amino acid sequences of the chemically modified peptides are determined by MALDI-TOF analysis; these amino acid sequences comprise all or part of a region on a protein that is chemically modified by the enzyme.
- the invention provides compositions and methods for extending sequence coverage in peptide-mass fingerprinting, comprising contacting the peptide with a MALDI matrix additive of the invention.
- the invention provides compositions and methods for inhibiting the formation of protein:ion adducts in a protein, comprising contacting the protein with a MALDI matrix additive of the invention.
- the invention provides compositions and methods for evaluating uncharacterized compounds and compositions for their potential as matrix additives of the invention (e.g., MS-compatible solubilizers, MS-compatible sorbents and/or MS-compatible buffers).
- matrix additives of the invention e.g., MS-compatible solubilizers, MS-compatible sorbents and/or MS-compatible buffers.
- kits comprising one or more containers comprising at least one of the MALDI matrix additives of the invention.
- kits can further comprise one or more kit components. Illustrative examples of such kits are provided herein (see especially Examples 11, 12 and 22).
- the invention is drawn to a solid support comprising or coated with a MS-compatible composition.
- the MS-compatible composition can be a MS-compatible solubilizer, or a composition comprising one or more MS-compatible non-volatile MALDI additives.
- the solid support can be in the form of a bead, a monolithic column or chip surface or the interior of chromatographic tubing. In some embodiments, the solid support is a MS target surface
- the invention is drawn to a method of coating a substrate, comprising contacting said substrate to one or more MS-compatible compositions.
- the MS- compatible composition can be a MS-compatible solubilizer, or a composition comprising one or more MS-compatible non-volatile MALDI additives.
- such methods involve electrospraying.
- Figure 1 SDS-PAGE of purified T. californica nAChR.
- Figure 2 MALDI-MS spectra for the peptide mass fingerprint of the nAChR delta (A), gamma (B), beta (C) and alpha (D) subunits.
- Figure 3 Amino acid sequences of the nAChR delta (A), gamma (B), beta (C) and alpha (D) subunits.
- Bold text signifies residues identified by MALD-TOF as described in the Examples; underlined text corresponds to transmembrane domains.
- FIG. 4 MALDI-TOF-MS analysis of the PMF for BSA.
- the spectra of trypsinized BSA without (A) or with (B) IX 1MB between m/z 825 and 1700 are shown.
- the numbers above each mass-ion identify the corresponding region of the amino acid sequence of BSA.
- the font color is coordinated with identified NA adducts.
- the spectra on the right are expanded views of the spectra between m/z 1190 and 1310.
- FIG. 5 MALDI-TOF-MS analysis of Bradykinin.
- Bradykinin m/z 998.58 was analyzed without 1MB in the presence of (A) 50 mM NaCl or (C) 50 mM KCl; the sodium adducts (m/z 1020.56, 1042.54) and potassium adduct (m/z 1036.55) are identified by green font.
- 0.4X EVIB 1 : 1 (v/v) was included in these experiments, in either (B) 50 mM NaCl or (D) 50 mM KCl, the signal from the adducts was reduced or eliminated.
- Figure 6 Plot of normalized intensity of + Na adduct versus concentration of MB.
- Figure 7 Plot of normalized intensity of + Na adduct versus laser intensity.
- Figure 8 MALDI-MS spectra of 100 fmol of BSA in (A) 50% acetonitrile/0.1%TFA (trifluoroacetic acid) and (B) in IX BLEND 1/0.7 M urea/0.7 M thiourea.
- Figure 9 Chromatographic separation of cytochrome P450 tryptic digest in the presence of Blend II monitored as a total ion count by ESI-MS.
- Figure 10 SDS-PAGE of acetone-preciptated proteins (myoglobin, BSA and nAChR) resuspended in distilled water (dH 2 O), NuPAGE buffer, or BLEND II with 4 M urea.
- acetone-preciptated proteins myoglobin, BSA and nAChR
- Figure 1 1 Recovery of nAChR from dialysis using BLEND II.
- Figure 12 Total ion chromatograph for a cytochrome P450 (SEQ ID NO: 8) 2D6 digest in BLEND II.
- Figure 13 In-gel digestion of nAChR in the presenced or absence of BLEND II.
- Figure 14 Spectra of mass standards in ordinary MALDI matrix versus matrix with silica additive. The top two spectra show MALDI-MS analyses of a calibrant mixture in (A) conventional alpha-cyano (CHCA) and (B) alpha-cyano:silica mixture. The bottom two spectra show MALDI-MS analyses of a calibrant mixture in the presence of 500 mM NaCl in (C) conventional alpha-cyano and (D) alpha-cyano: silica mixture.
- CHCA conventional alpha-cyano
- D alpha-cyano
- Figure 15 Spectra of a tryptic digest of Ovalbumin ( 1 pmol) analyzed by MALDI-MS using (A) a conventional CHCA ( ⁇ C) matrix and (B) Maxlon AC, in which silica is present.
- Figure 16 MALDI-MS analysis of tryptic digest of beta-galactosidase in (A) conventional CHCA (alpha-cyano) and (B) Maxlon AC, in which silica is present.
- Figure 17 Spectra of tryptic digests of 100 fmol of beta-galactosidase in 500 mM NaCl in (A) conventional CHCA ( ⁇ C) matrix or (B) Maxlon AC, in which silica is present.
- Figure 19 MALDI MS spectra of intact proteins (insulin, ubiquitin and cytochrome-c) co-spotted with SA only (Al -A3, B1-B3) or co-spotted with SA/MES (Maxlon SA) (C1-C3). (A, B, C, 1, 2 and 3 are as described for Figure 13).
- Figure 20 Analysis of a HMW standard (159,081 Da) using (A) sinapinic acid dissolved in 0.1% TF A/50% ACN and (B) sinapinic acid dissolved in Maxlon SA.
- Figure 21 Chemical structures of (A) sinapinic acid and MES and (B) MES, MOPS, MOPSO and MOBS.
- Figure 22 The silica resin of Maxlon AC tested under different storage conditions and analyzed by MALDI-MS of InvitroMass LMW CaI 2 (1 :200 in 100 mM NaCl).
- Figure 23 Matrix solution (0.5 ⁇ L) was co-spotted with 0.5 ⁇ L of 100 mM NaCl solution onto a stainless steel MALDI target plate (A) without or (B) silica.
- Figure 24 Spectra resulting from MALDI analysis of the target surfaces shown in Figure 18 gathered by random scanning of (A) the plate with silica and (B) the plate lacking silica.
- Analyte The terms “analyte” and “molecule of interest” are used interchangeably herein to indicate a molecule that one wishes to detect, quantify or otherwise examine or study. That is, the use of the term term “analyte” herein is not limiting to only determining the type or amount of a molecule of interest; rather, it encompasses other observations regarding e.g., ligand:ligand interactions and conformational change of molecules.
- Bead A spheroidal solid support.
- a bead can but need not be hollow, or can comprise openings from the outer surface of the bead that lead to one or more internal surfaces.
- the external and/or internal surfaces of a bead can be coated with a molecule having one or more useful properties.
- a bead coated with a binding moiety e.g., an antibody
- a sample that contains the ligand e.g., the antigenic target of the antibody
- the bead will bind and retain the ligand (antigen).
- the bead is washed or otherwise treated to remove undesirable contaminants, and the ligand, a molecule of interest, is eluted from the bead.
- a population of beads is placed in a hollow container within which flows a fluid containing a molecule of interest, and the beads' surfaces are coated with a binding moiety, or an enzyme.
- a molecule of interest which is a substrate for a given enzyme, is contacted with beads coated with that enzyme, and the products of the chemical (enzymatic) reaction are generated.
- a sample can be contacted with a population of beads by fluid passage through a bead- filled container (e.g., a column) followed by optional washes and elution, or by preparing a mixture of beads and sample, which is then centrifuged to make the beads form a pellet, followed by optional washes and elution.
- a bead- filled container e.g., a column
- Biomolecule encompasses any molecule produced by a living organism or a fragment thereof.
- biopolymer as used herein means any polymeric molecule produced by a living organism or a fragment thereof. Either type of molecule can be a polypeptide, a protein, a nucleic acid, a polynucleotide, a carbohydrate, a lipid, a polysaccharide, or a fragment or derivative thereof.
- Chaotrope The term “chaotrope” as used herein refers to a chemical agent that denatures proteins. Exemplary chaotropes include urea, thiourea and guanidine hydrochloride. The terms “chaotrope”, “denaturing agent”, and “denaturant” are used interchangeably herein.
- Coat refers to a layer of a substance on the surface of a solid support, which can be, for example, a bead; all or a portion of a well in a microtiter plate; the inner surface of a container; and the like.
- Colloid As used herein, a "colloid" is a mixture composed of particles (the dispersed phase) suspended in a medium (a continuous mobile phase), having properties between those of a solution and a fine suspension. Colloidal particles generally have at least one dimension in the range of from 1 (or about 1) run to 100 (or about 100) micrometers, particularly from 10 (or about 10) nra to 50 (or about 50) micrometers, particularly from 10 (or about 10) nra to 10, 15 or 20 (or about 10, 15 or 20) micrometers. Unless otherwise specified, as used herein "colloid” refers to the colloid mixture per se and the particles without the mobile phase (i.e., dried particles). The term “colloid” also encompasses sols, slurries, colloidal suspensions and resins.
- colloidal suspension As used herein, a "colloidal suspension” (a.k.a. colloidal solution) is a thermodynamically stable colloid comprised of particles suspended in a liquid. Typically, a colloidal suspension can be observed to have the Tyndall Effect, in which the reflection of a light beam passing through a colloid identifies the presence of suspended particles.
- Cross-linker The terms "cross-linker” and “cross-linking agent” are used interchangeably herein and are intended to refer to a typically bifunctional (two-armed) chemical linker that can be added to a mixture of molecules to form covalent linkages between two or more molecules.
- Such bifunctional cross-linkers can be homobifunctional (wherein both "arms” of the linker are the same chemical moiety) or heterobifunctional (wherein each of the two “arms” is a different chemical moiety than the other).
- Reactive groups that can be targeted using a cross-linker include primary amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids. Many cross-linkers are described and made commercially available by Pierce Biotechnology, Inc. (Rockford, IL).
- Detectablv labeled The terms "detectably labeled” and “labeled” are used interchangeably herein and are intended to refer to situations in which a molecule (e.g., a nucleic acid molecule, protein, nucleotide, amino acid, and the like) have been tagged with another moiety or molecule that produces a signal capable of being detected by any number of detection means, such as by instrumentation, eye, photography, radiography, and the like.
- a molecule e.g., a nucleic acid molecule, protein, nucleotide, amino acid, and the like
- molecules can be tagged (or "labeled") with the molecule or moiety producing the signal (the "label” or “detectable label”) by any number of art-known methods, including covalent or ionic coupling, aggregation, affinity coupling (including, e.g., using primary and/or secondary antibodies, either or both of which may comprise a detectable label), and the like.
- Suitable detectable labels for use in preparing labeled or detectably labeled molecules in accordance with the invention include, for example, radioactive isotope labels, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels, and others that will be familiar to those of ordinary skill in the art.
- domain and "protein domain” are used interchangeably herein to refer to a relatively small (i.e., ⁇ about 150 amino acids) globular unit that is part of a protein.
- a protein may comprise two or more domains that are linked by relatively flexible stretches of amino acids.
- a given domain may be largely or wholly responsible for carrying out functions that are normally carried out by the intact protein.
- domains that have been determined by in vitro manipulations of protein molecules, it is understood in the art that a "domain” may also have been identified in silico, i.e, by software designed to analyze the amino acid sequences encoded by a nucleic acid in order to predict the limits of domains. The latter type of domain is more accurately called a "predicted” or “putative” domain but, in the present disclosure, the term domain encompasses both known and predicted domains unless stated otherwise.
- Hvdrophilic The terms “hydrophilic” and “lipophobic” are used interchangeably herein and refer to compounds and substances that tend to dissolve in, mix with or be wetted by, water. Hydrophilic or lipophobic species, or hydrophiles, tend to be electrically charged and polar, and thus preferring other charged and polar solvents or molecular environments. Non-limiting examples of hydrophilic molecules include lipids and hydrophilic proteins.
- Hydrophobic The terms “hydrophobic” and “lipophilic” are used interchangeably herein and refer to compounds and substances that tend to not dissolve in, mix with or be wetted by, water. Hydrophobic or lipophilic species, or hydrophobes, tend to be electrically neutral and nonpolar, and thus preferring other neutral and nonpolar solvents or molecular environments. Non-limiting examples of hydrophobic molecules include alkanes, oils, fats, lipids and hydrophobic proteins.
- Ligand refers to a small molecule that binds to a larger macromolecule.
- ligands are a synthetic compound, such as a drug or drug candidate (lead compound), or a biomolecule, e.g., antibody, an agonist, an antagonist, an allosteric modulator, a phospholipid, cholesterol, a fatty acid, a steroid, a hormone, a volatile anesthetic, a fluxing ion, an ion cofactor or modulator, or combinations thereof.
- Molecule The term "molecule” has its normal scientific meaning herein, but also includes molecular complexes.
- Molecular Complex A type of molecule (as that term is used herein) that consists of two or more molecules that are at least partially bound to each other due to non-chemical interactions.
- examples of some molecular complexes of particular interest include protein:nucleic acid complexes, e.g., recombination complexes, topoisomerase complexes, RNA or DNA polymerase holoenzymes, ribosomes, and the like.
- Monomer refers to the unimolecular form of a molecule that can achieve a multimeric form.
- Multimer refers to a complex or compound formed by the assembly of 2 or more monomers.
- a multimer is a protein complex that is formed of two or more copies of the same polypeptide, e.g., the homodimeric nucleoid-associated protein HBsu of Bacillus subtilis.
- Another example is a lipids that can assemble into aggregate structures such as micelles and bilayers.
- Non-volatile As used herein, the phrase “non- volatile” refers to a composition that is not readily vaporizable at a relatively low temperature, especially room temperature.
- nucleic acid molecule refers to a sequence of contiguous nucleotides (riboNTPs, dNTPs, ddNTPs, or combinations thereof) of any length.
- a nucleic acid molecule may encode a full-length polypeptide or a fragment of any length thereof, or may be non-coding.
- nucleic acid molecule and polynucleotide may be used interchangeably and include both RNA and DNA.
- the term "of interest” is used to indicate a particular object or process that one wishes to detect, identify, quantify, determine or monitor the activity or properties of, and/or otherwise observe. Unless otherwise indicated, as used herein the term “molecule of interest” is synonymous with “analyte”.
- Polypeptide refers to a sequence of contiguous amino acids of any length, i.e., a linear molecule composed of two or more amino acids linked by covalent (peptide) bonds.
- the terms "peptide,” “oligopeptide,” or “protein” may be used interchangeably herein with the term “polypeptide.”
- the term “protein” includes polypeptides as well as protein complexes formed of 2 or more polypeptides.
- Purified refers to a compound that has been separated from at least 50% or about 50% of undesirable elements in a mixture containing the compound.
- Resin refers to the polymeric base (which may be chemically modified, e.g., cross-linked to one or more other substances) of some ion- exchange materials used in chromatography.
- the polymeric base may be, but need not be, polystyrene.
- sample refers to any composition that is subject to analysis. Typically, a sample comprises, or is suspected of comprising, an analyte of interest.
- Separated refers to a compound that has been physically separated from at least one other element in a mixture containing the compound.
- Slurry refers to a thin mixture of a liquid, especially water, and any of several finely divided substances, such as beads, cement, plaster of Paris, or clay particles.
- Solid support means a non-gaseous, non-liquid, solid or semi-solid material having a surface.
- a solid support can be a flat surface constructed, for example, of glass, silicon, metal, plastic or a composite; or can be in the form of a bead such as a silica gel, a controlled pore glass, a magnetic or cellulose bead; or can be a pin, a monolithic column, a chip surface, or the interior of chromatographic tubing including an array of pins suitable for combinatorial synthesis or analysis.
- Solubilizer The terms “solubilizer” and “solubilizing agent” are used interchangeably herein and refer to any compound or mixture of compounds that enhances the solubility of a hydrophobic compound.
- Solution As used herein, a “solution” is a homogeneous mixture that is a single- phase mixture composed of a solute and a solvent.
- the dissolved particles (solutes) are small molecules and ions between 1 A and 100 A in diameter.
- Surfactants Surface active molecules or compositions; also known as wetting agents. Surfactants are used to provide detergency and emulsification.
- the term “surfactant” encompasses detergents as well as non-detergents (e.g., non-detegent sulfobetaines, a.k.a. NDSBs).
- Suspension As used herein, a "suspension" is a two-phase mixture composed of a dispersed and continuous phase. The particles of the dispersed phase are generally larger than about 10,000 A to about 100,000 A (i.e., from about 1 micrometer to about 10 micrometers) thick in at least one dimension (e.g., length, width, height, depth diameter.
- the invention is drawn to reagents that can be used in MS that improve the solubility of analytes during sample processing (for any type of MS analysis) or in MALDI sample-matrix formulations, increase the signal-to-noise ratio of mass spectra, reduce the size of adduct cluster peaks in mass spectra, increase the analyzable surface area of a MALDI sample, or improve the stability of an anal yte: matrix crystal used in MALDI-MS.
- the invention is drawn to MALDI matrix additives.
- Such MALDI matrix additives comprise one or more substances selected from the group consisting of a MS-compatible solubilizer, a MS-compatible sorbent, and a MS-compatible buffer.
- a MS-compatible solubilizer for example, a MS-compatible solubilizer, a MS-compatible sorbent, and a MS-compatible buffer.
- the invention is not limited in use to MALDI MS or with MALDI matrices.
- the solubilizers, buffers, and reagents used in the invention can find use in other types of MS analysis, including LC/MS.
- an MS-compatible reagent of the invention comprises one or more MS-compatible solubilizers, one or more MS-compatible sorbents, and/or one or more MS-compatible buffers.
- the invention is drawn to compositions and kits comprising one or more MS reagents of the invention, and methods of making and using such compositions and kits.
- MS target molecule of "analyte” refers to a molecule of interest that is being studied using MS.
- MS target molecules include peptides; proteins; protein:protein complexes; protein:DNA complexes; oligonucleotides; nucleic acids, such as DNA and RNA; nucleic acid:nucleic acid complexes; oligosaccharides; lipids, including phospholipids; synthetic polymers; small organic molecules; and complexes of any of the above.
- any of these molecules can be from a biological source (“biomolecules”) or from in vitro chemical synthesis (“synthetic molecules”)-
- the molecule is a hydrophobic molecule, such as a lipid or a hydrophobic protein (e.g., a membrane protein), but the invention is applicable to hydrophilic molecules (e.g., soluble proteins) as well.
- MS reagents of the invention are not necessarily present in the analyte:matrix crystals placed on a MALDI target surface. They are, however, present for at least part of the MALDI analysis procedure and can thus be added at one or more various points, including without limitation: during sample preparation or sample processing procedures; during preperation of the matrix molecules; during formation of analyte:matrix crystals; during washing of the target surface; etc.
- sample processing procedures refers to procedures involving preparing a sample for MALDI-TOF MS, including without limitation (a) analyte isolation; (b) sample processing; (c) mixing of the analyte, matrix and optional additives; (d) co-precipitation of analyte: matrix to produce analyte:matrix crystals; (e) deposition of analyte:matrix crystal onto a MS target surface; and (f) washing analyte:matrix crystals in situ.
- the MS-compatible reagents of the invention can be partially or totally removed at any time after their addition.
- the additives can be combined with any other component(s) in any fashion and in any appropriate order.
- the MS-compatible reagents provided herein are used as matrix additives that are present in a MALDI matrix crystal during MS analysis.
- the additive can be combined with a matrix and a sample comprising an analyte as those two componenents are mixed, or "pre-mixed" with matrix or analyte before matrix and analyte are combined.
- matrix molecules can be dissolved in a diluent that comprises one or more matrix additives of the invention.
- crystals comprising the additives are also part of the claimed invention.
- the matrix additive can be a MS-compatible solubilizer, such as one or more sulfobetaines, one or more non-detergent sulfo-betaines, a MS-compatible sorbent, such as silica, and/or a MS-compatible buffer of the invention.
- a MS-compatible solubilizer such as one or more sulfobetaines, one or more non-detergent sulfo-betaines, a MS-compatible sorbent, such as silica, and/or a MS-compatible buffer of the invention.
- compositions and methods of the invention can be applied to any appropriate analyte.
- the analyte can be a hydrophilic molecule or a hydrophobic molecule.
- the analyte can be a protein complex, or a molecule selected from the group consisting of other proteins, peptides, DNA, RNA, oligonucleotides, nucleic acids, oligosaccharides, polysaccharides, lipids, phospholipids, synthetic polymers, small organic molecules, and complexes or combinations of any of the above.
- the invention provides compositions comprising solubilizing agents (solubilizers) that are not, unlike other solubilizers, largely incompatible with mass spectrometry (i.e., they are MS-compatible); MS-compatible matrix additives; mixtures, including crystals, comprising MS matrix and/or analyte molecules and one or more MS- compatible compositions (e.g., a MS-compatible solubilizer or a MS-compatible matrix additive); and solutions comprising one or more MS-compatible compositions.
- the MS-compatible solubilizer is not removed from a sample or analyte during MS, but is present during mass spectrometry.
- the provided MS-compatible solubilizers do not have deleterious effects on mass spectra of analytes.
- the MS-compatible solubilizers can include without limitation detergents or surfactants. Blends of solubilizers are included, in which the blends can include one or more detergents or one or more surfactants, and one one or more detergents in combination with one or more surfactants.
- the solubilizer blends can optionally further include buffers, chaotropic agents, salts, or other chemical entities.
- a MS-compatible solubilizer of the invention can be a MS-compatible detergent, a MS-compatible non-detergent, or combinations thereof. More specifically, a MS-compatible solubilizer of the invention comprises one or more components selected from the group consisting of (a) one or more MS-compatible detergents, wherein at least one of the detergents is at a concentration that is at least about 5% of its CMC when in solution with the analyte prior to crystal formation, more preferably at a concentration that at least about 75% of its CMC when in solution with the analyte prior to crystal formation; and (b) one or more MS-compatible non-detergent surfactants, wherein an effective amount of said MS- compatible solubilizer has one or more of the following characteristics when used in mass spectrometry studies: (i) it improves the solubility of an analyte by at least about 5% during one or more sample processing procedures, (ii) it improves the solubility of an analyte by at least about
- MS-compatible solubilizing compositions include at least one detergent that is present at a concentration close to its CMC. In some preferred embodiments, MS-compatible solubilizing compositions include at least one detergent that is present at a concentration that is at or above its CMC.
- compositions can included two or more MS-compatible detergents, each of which is at a concentration of at least about 75% of its CMC when in solution with an analyte prior to mass spectrometry.
- the compositions can included two or more MS-compatible detergents, each of which is at a concentration of close to its CMC when in solution with an analyte prior to mass spectrometry.
- the compositions can included two or more MS- compatible detergents, each of which is at a concentration at or above its CMC when in solution with an analyte prior to mass spectrometry.
- analytes are prepared for MS-MALDI analysis by contacting a sample comprising analyte molecules with molecules of a MALDI matrix material.
- the analyte and matrix molecules co-precipitate to form what is called an "analytermatrix crystal" herein.
- the crystal which is formed on a MALDI target surface, is subject to pulses of laser irradiation and, as a result, the analyte molecules are ionized.
- the ions are subject to further analysis, e.g., time-of-flight (TOF) analysis.
- TOF time-of-flight
- an effective amount of a MS-compatible solubilizer of an invention increases the signal-to-noise ratio from at least about 5% to about 100-fold.
- the MS-compatible solubilizer is used at least at a concentration at which it is effective to improve the solubility of the molecule of interest by about 10% during analyte:matrix crystallization and/or during laser exposure in MALDI-MS, preferably resulting in an at least about 10% increase of signal-to-noise ratio.
- An MS-compatible solubilizer can also be used to enhance solubility of one or more analytes using other types of MS, in particular LC/MS.
- an MS-compatible solubilizer can allow for better yield and purification of proteins separated by, for example, HPLC, RP HPLC, capillary electrophoresis, or liquid or gel phase isoelectric focusing prior to MS analysis.
- HPLC high-LC
- RP HPLC RP HPLC
- capillary electrophoresis or liquid or gel phase isoelectric focusing prior to MS analysis.
- a MS-compatible solubilizer of the invention comprises one or more surfactants.
- a surfactant may be a detergent or a non-detergent surfactant, or combinations thereof.
- a solubilizer that is a mixture (blend) of surfactants can be MS-compatible even if individual surfactants are not. In some embodiments, however, MS-compatible surfactants are preferred.
- Non-limiting examples of non-detergent surfactants include the non-detergent sulfobetaines (NDSBs).
- NDSBs are zwitterionic compounds that have a sulfobetaine hydrophilic group and a short hydrophobic group. They cannot aggregate to form micelles, and NDSBs are thus not considered detergents.
- NDSBs that can be used in the invention include without limitation those listed in Table 1.
- a solubilizer composition of the invention comprises at least one NDSB.
- the concentration of an NDSB in a solubilizer composition can vary, for example such that the concentration of the NDSB when it contacts a sample or analyte is from about 10 micromolar to about 2M, such as from about 100 micromolar to about IM, or from about 1 mM to about 800 M. In preferred embodiments, the concentration of an NDSB when it contacts a sample or analyte is at least about 5mM, such as from about 10 mM to about 600 mM, or from about 20 micromolar to about 400 mM, or from about 50 mM to about 300 mM.
- NDSBs NON-LIMITING EXAMPLES OF NON-DETERGENT SULFOBETAINES
- a detergent is a compound, or a mixture of compounds, the molecules of which have two distinct regions: one that is hydrophilic, and readily dissolves in water, and another that is hydrophobic, with little (if any) affinity for water.
- a detergent is thus an amphipathic surface-active molecule, i.e., one type of surfactant. Unlike non-detergent surfactants, detergents can form micelles.
- Non-ionic detergents are molecules that do not ionize in aqueous solutions. Ionic detergents can be divided into those having cationic (positively charged) and anionic (negatively charged) detergents.
- a Zwitterion German for "hybrid ion" is a neutral compound having electrical charges of opposite sign, delocalized or not on adjacent or nonadjacent atoms. Zwitterionic compounds have no uncharged canonical representations, and can behave like an acid or a base, depending on conditions.
- Preferred detergents for use in MS-compatible solubilizer compositions include nonionic detergents and zwitterionic detergents.
- nonionic detergents used in MS compatible solubilizer compositions can be glycopyranosides, and include but are not limited to nonionic detergents having glucose, maltose, or sucrose moieties.
- Preferred zwitterionic detergents for use in MS compatible solubilizer compositions are sulfobetaine detergents, for example, SB or ASB detergents (e.g., SB-8, SB- 10, SB-12, SB-14, SB-16, ABS-C80).
- concentration of the detergents used can vary. Methods are provided herein for testing the effectiveness of detergent formulations in improving spectra.
- at least one of the detergents used in a solubilizer for MS is contacted with a sample or analyte near or above its CMC.
- Anionic detergents include without limitation: [00126] Glycochenodeoxycholic acid sodium salt; Glycocholic acid hydrate, synthetic; Glycocholic acid sodium salt hydrate; Glycodeoxycholic acid monohydrate; Glycodeoxycholic acid sodium salt; Glycolithocholic acid 3-sulfate disodium salt; and Glycolithocholic acid ethyl ester;
- Taurochenodeoxycholic acid sodium salt Taurodeoxycholic acid sodium salt monohydrate; Taurohyodeoxycholic acid sodium salt hydrate; Taurolithocholic acid 3-sulfate disodium salt; and Tauroursodeoxycholic acid sodium salt;
- Cationic detergents include without limitation:
- Benzyldimethylhexadecylammonium chloride Benzyldimethyltetradecylammonium chloride; Benzyldodecyldimethylammonium bromide; and Benzyltrimethylammonium tetrachloroiodate; Dimethyldioctadecylammonium bromide;
- Thonzonium bromide and Trimethyl(tetradecyl)ammonium bromide.
- Non-ionic detergents include without limitation:
- Span® detergents including without limitation Span® 20; Span® 40; Span® 60; Span® 65; Span® 80; and Span® 85;
- Tergitol detergents including without limitation Tergitol, Type 15-S-12; Tergitol, Type 15-S-30; Tergitol, Type 15-S-5; Tergitol, Type 15-S-7; Tergitol, Type 15-S-9; Tergitol, Type NP-IO; Tergitol, Type NP-4; Tergitol, Type NP-40; Tergitol, Type NP-7; Tergitol, Type NP-9; Tergitol, Type TMN-IO; and Tergitol, Type TMN-6;
- Hexaethylene glycol monododecyl ether Hexaethylene glycol monohexadecyl ether; Hexaethylene glycol monooctadecyl ether; and Hexaethylene glycol monotetradecyl ether;
- Octaethylene glycol monodecyl ether Octaethylene glycol monododecyl ether; Octaethylene glycol monohexadecyl ether; Octaethylene glycol monooctadecyl ether; and Octaethylene glycol monotetradecyl ether; Octyl-b-D-glucopyranoside; octyl-beta-D-1- thioglucopyranoside;
- Pentaethylene glycol monodecyl ether Pentaethylene glycol monododecyl ether; Pentaethylene glycol monohexadecyl ether; Pentaethylene glycol monohexyl ether; Pentaethylene glycol monooctadecyl ether; and Pentaethylene glycol monooctyl ether;
- Tetraethylene glycol monodecyl ether Tetraethylene glycol monododecyl ether; and Tetraethylene glycol monotetradecyl ether;
- Triethylene glycol monodecyl ether Triethylene glycol monododecyl ether; Triethylene glycol monohexadecyl ether; Triethylene glycol monooctyl ether; and Triethylene glycol monotetradecyl ether;
- APO-10 As well as APO-10; APO- 12; Bis(polyethylene glycol bis[imidazoyl carbonyl]); Cremophor® EL; Decaethylene glycol monododecyl ether; Tyloxapol; and n-Undecyl-beta- D-glucopyranoside; Igepal CA-630; Methyl-6-O-(N-heptylcarbamoyl)-a-D-glucopyranoside; Nonaethylene glycol monododecyl ether; N-Nonanoyl-N-methylglucamine; NP-40; propylene glycol stearate; Saponins, e.g., Saponin from Quillaja bark; and Tetradecyl-b-D- maltoside.
- Zwitterionic detergents include without limitation:
- Zwittergent® detergents including without limitation Zwittergent® 3-08 («- Octyl-N,N-dimethyl-3-ammonio-l-propanesulfonate); Zwittergent® 3-10 (n-Decyl-N,N- dimethyl-3-ammonio-l-propanesulfonate); Zwittergent® 3-12 (3-Dodecyl- dimethylammonio-propane-1 -sulfonate); Zwittergent® 3-14 (w-Tetradecyl-N,N-dimethyl-3- ammonio-1-propanesulfonate); and Zwittergent® 3-16 ( «-Hexadecyl-N,N-dimethyl-3- ammonio-1-propanesulfonate) [all of these Zwittergents are commercially available from EMD Biochemicals/Calbiochem, San Diego, CA]; [00148] 3-(Decyldimethylammonio)propanesulfonate)
- Sulfobetaine detergents including sulfobetaine SB8, sulfobetaine SBlO, sulfobetaine SB 12, sulfobetaine SB 14, sulfobetaine SB 16, and 4-n-Octylbenzoylamido- propyl-diniethylammoniosulfobetaine (ASB-C80);
- Preferred MS-compatible detergents and surfactants include alkyl glycosides, sulfobetaines, non-detergent sulfobetaines, and bile acids.
- Preferred detergents for use in the invention are MS-compatible meaning, in general, that they have no characteristics that interfere with a MALDI-TOF MS analysis of choice.
- An "MS-compatible" compound meets these characteristics: (1) it does not significantly interfere with the ionization efficiency of the analyte; (2) it does not form adducts to the protein or peptide that would interfere with mass determination; (3) it does not interfere w/ matrix crystal formation; and (4) it does not interfere with sample preparation.
- Detergents that can act as solubilizers for hydrophobic proteins are preferred.
- CMC's for detergents can be found in CRC Guide for Surfactants and Lipids [00154] Provided herein is a method for determining whether a compound such as a micelle-forming detergent or a surfactant-like compound is mass-spectroscopy compatible.
- This method provides another embodiment of the present invention.
- the method is illustrated in Example 1 , herein.
- An important characteristic of detergents and surfactants useful for practicing the present invention is the amount and type of aggregate structures present or formed during methods of MS and/or preparing samples for MS.
- monomers are preferred and aggregate structures, include without limitation liposomes and micelles, are less desirable.
- Detergent monomers assemble into aggregates called micelles, wherein the hydrophobic and hydrophilic moieties are exposed to the micelle interior and the aqueous environment, respectively.
- CMC critical micelle concentration
- compositions of the present invention are drawn to a composition comprising a detergent at a concentration that is at a concentration that is less than its CMC, including without limitation 99% or about 99%, 95% or about 95%, 90% or about 90%, 80% or about 80%, 75% or about 75%, 60% or about 60%, 50% or about 50%, 40% or about 40%, 30% or about 30%, 20% or about 20%, 10% or about 10%, and 1% or about 1% of its CMC.
- a formulation for an MS-compatible solubilizer includes at least one component at a concentration which is above its CMC.
- compositions of the present invention are drawn to a composition comprising a detergent at a concentration that is at a concentration that is approximately equal (100%) to its CMC, or at a concentration greater than its CMC, including without limitation 101% or about 101%, 110% or about 110%, 125% or about 125%, 150% or about 150%, 175% or about 175%, 2X or about 2X, 3X or about 3X, 4X or about 4X, 5X or about 5X, 6X or about 6X, 7X or about 7X, 8X or about 8X, 9X or about 9X, 1OX or about 10X, 25X or about 25X, IOOX or about 10OX, of its CMC.
- the Molecular Weight (MW) of a specific micelle can be calculated by multiplying the MW of a monomer of the detergent times the micelle's aggregation number.
- the MW of a particular micelle is of interest in some aspect of the invention, including dialysis. More specifically, a dialysis membrane can have a molecular weight cut-off; that is, only molecules below a certain MW can freely pass through the membrane.
- the MW of a micelle can be much larger than that of the detergent monomer of which it is composed.
- Table 2 shows characteristics, including MW, Aggregation Number and Micellar MW of several representative but non-limiting detergents. TABLE 2: CRITICAL MICELLE CONCENTRATION (CMC) AND MOLECULAR WEIGHT OF MICELLES FOR SEVERAL DETERGENTS
- Membrane proteins of interest are often solubilized by the presence of a detergent, including without limitation the detergents presented in Table 3.
- Detergents and non-detergent surfactants include without limitation those provided in the examples herein.
- lipids are, like detergents, amphipathic surface-active molecules.
- lipids are any of a variety of oily or greasy organic compounds found as major structural components of living cells; they are insoluble in water but soluble in organic solvents such as alcohol and ether, and include the common fats, cholesterol and other steroids, phospholipids, sphingolipids, waxes, and fatty acids.
- lipids are fatty acid esters, a class of relatively water-insoluble organic molecules. There are three forms of lipids: phospholipids, steroids. and triglycerides.
- Lipids consist of a polar or hydrophilic (attracted to water) head and one to three nonpolar or hydrophobic tails. Since lipids have both functions, they are called amphophilic.
- the hydrophobic tail consists of one or two (in triglycerides, three) fatty acids. These are unbranched chains of carbon atoms (with the correct number of H atoms), which are connected by single bonds alone (saturated fatty acids) or by both single and double bonds (unsaturated fatty acids). The chains are usually 14-24 carbon groups long.
- the hydrophilic head is from one of three groups: (1) glycolipids, whose heads contain an oligosaccharide with 1-15 saccharide (sugar) residues; (2) phospholipids, whose heads contain a positively charged group that is linked to the tail by a negatively charged phosphate group; and (3) sterols, whose heads contain a planar steroid ring, for example, cholesterol.
- Lukas et al. (Anal Biochem. 301 :175, 2002) state that the efficiency of extraction of a hydrophobic protein (nAChR) from membranes was not markedly different for the detergents tested, but quality and signal size of mass spectra were influenced by the composition and concentration of the detergents, as well as the concentration of protein and MALDI matrix composition. Lukas et al. state that their best spectra were obtained for samples solubilized in Triton X-100 and assayed by use of a sinapinic acid matrix.
- nAChR hydrophobic protein
- the MS-compatible detergent can be at a concentration of at least about 25%, about 50%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or greater than about 100%, for example about 110%, about 125%, about 150%, about 200%, about 300%, about 400%, or about 500% of its critical micelle concentration (CMC).
- CMC critical micelle concentration
- the MS-compatible detergent can be at a concentration of at least 75%, 80%, 85%, 90%, 95%, 100%, or greater than 100%, for example 110%, 125%, 150%, 200%, about 300%, about 400%, or about 500% of its CMC when it contacts a sample that contains one or more analytes and subsequently the sample can be diluted, for example, to bring the concentration of the solubilizer to a concentration below its CMC, prior to performing mass spectrometry.
- a matrix compound can be added to a sample or analyte prior to adding an MS compatible solubilizer to a sample or analyte or after adding an MS solubilizer to a sample or analyte.
- a matrix compound added to a sample after adding an MS solubilizer to a sample can be added before or after any dilution of the sample/solubilizer solution that may be performed.
- the MS-compatible detergent is preferably at a concentration at or below its CMC, but this is not a requirement of the present invention.
- the concentration of an MS compatible detergent can be at least about 1%, at least about 5%, about 10%, about 25%, about 30% about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% about 100%, or greater than 100%, for example 110%, 125%, 150%, or 200% of its critical micelle concentration (CMC), hi certain preferred embodiments, the MS-compatible solubilizer is at a concentration of less than 100%, of its CMC immediately prior to crystal formation and mass spectrometry analysis.
- CMC critical micelle concentration
- an MS-compatible solubilizer of the invention comprises one or more MS-compatible detergents or non-detergent surfactants, or blends (mixtures) thereof.
- MS-compatible detergents or non-detergent surfactants or blends (mixtures) thereof.
- a MS-compatible solubilizer of the invention comprises one or more chemical compounds identified by structure and chemical formula herein. These include without limitation alkyl glycosides, sulfobetaine detergents, non-detergent sulfobetaines (NDSBs), bile acids and Rabilloud detergent variants, hi particular, see ExamplelS, Illustrative Detergents, Non-Detergents and Other Compositions for MS- Compatible Solubilizers.
- a MS-compatible solubilizer of the invention comprises one or more MS-compatible non-detergent surfactants.
- the MS-compatible non-detergent surfactant is a compound that is capable of forming bonds with a hydrophobic portion of a molecule and forming bonds with hydrophilic solvent molecules as well, thus preventing aggregation and precipitation of the hydrophobic molecule.
- non-detergent surfactants enhance the solubility of hydrophobic molecules by simultaneously forming bonds with the hydrophobic molecule and the surrounding solvent molecules but, unlike detergents, lack the ability to aggregate into micellar structures.
- Optimal concentrations of solubilizers in a formulation can be determined empirically using tests set forth in the example for the effects of matrix additives on signal-to- noise ratio of mass spectra, on the size of adduct cluster peaks in mass spectra, on the analyzable surface area of a MALDI sample, or on the stability of an analyte:matrix crystal used in MALDI-MS.
- Non-limiting examples of non-detergent surfactants are non-detergent sulfobetaines (NDSBs), such as NDSB-195, NDSB-201, NDSB-211, NDSB-221, NDSB- 223, and NDSB-256.
- NDSBs non-detergent sulfobetaines
- a MS-compatible solubilizer of the invention comprises an NSBD at a concentration of from about 5 mM to about 1 M, or from about 10 mM to about 0.8 M, or from about 50 mM to about 700 mM, or from about 10OmM to about 600 mM.
- NSBD-201 can be present in a solubilizer at a concentration of from about 125 mM to about 500 mM, for example 250 mM to 500 mM, and can be present in a solution with analyte to be analyzed by MS at a concentration of from about 10 mM to about 500 mM, preferably between about 20 mM and about 400 mM, when in solution with the analyte immediately prior to crystal formation.
- an MS-compatible solubilizer comprises NSBD-201 at a concentration of about 250 mM when in solution with the analyte immediately prior to crystal formation and mass spectrometry analysis.
- an MS-compatible solubilizer comprises NSBD-201 at a concentration of about 25 mM when in solution with the analyte immediately prior to crystal formation and mass spectrometry analysis.
- a MS-compatible solubilizer of the invention comprises one or more organic co-additives.
- organic co-additives include without limitation phospholipids, fatty acids, steroid compounds and organic solvents.
- An MS-compatible solublizer solution can comprise one or more buffers, acids, bases, or salts, such as, for example, ammonium bicarbonate at a concentration of from 10 to 100 mM.
- a MS-compatible solubilizer of the invention comprises one or more of the specific mixtures of MS-compatible detergents and/or MS-compatible non-detergent surfactants disclosed herein.
- Various combinations can be empirically tested. For example, a combination of ASB-C80, Octyl-beta-D-1-thioglucopyranoside, n- Dodecanoylsucrose, and SB 14 can be used.
- the concentrations of the detergent components can be such that one or more detergents is contacted with an analyte at a concentration above its CMC.
- Examples of commercially available MS-compatible solubilizers that can be used in this and other methods and compositions of the present invention are Invitrosol A, Invitrosol B and Invitrosol LC (Invitrogen, Carlsbad, CA).
- the invention also provides stock solutions of MS-compatible solubilizers.
- a stock solution is one that must be diluted to achieve a desired final working concentration.
- a 5x solution of a MS-compatible solubilizer of the invention can comprise ASB-C80 at from about 0.05 to about 10 mM, and preferably from about 0.1 to about 0.5 mM; Octyl-beta-D-1-thioglucopyranoside at from about 10 to about 500 mM and preferably from about 20 mM to about 250 mM; n-Dodecanoylsucrose from about 0.5 to about 20 mM; and SB 14 at about 0.1 to about 10 mM and preferably from about 0.2 mM to about 5 mM.
- An exemplary 5x solution of a MS-compatible solubilizer of the invention comprises ASB-C80 at 0.125 mM; Octyl-beta-D- 1 -thioglucopyranoside at 50 mM; n- Dodecanoylsucrose at 3.8 mM; and SB14 at 1 mM.
- the exemplary solubilizer can have a concentration of ASB-C80 or from about 0.01 to about 0.5 mM, more preferably from about 0.02 mM to about 0.1 mM, or in one embodiment, about 0.025 mM; a concentration of Octyl-beta-D- 1- thioglucopyranoside of from about 1 mM to about 50 mM, more preferably from about 5 mM to about 25 mM, or in one embodiment, about 10 mM; a concentration of n- Dodecanoylsucrose from about 0.1 to about 10 mM, more preferably from about 0.5 to about 5 mM, or or in one embodiment, about 0.76 mM; and a concentration of SB-14 of from about 0.05 mM to about 1 mM, more preferably from about 0.1 mM to about 0.5 mM, or about 0.2 mM.
- the solubilizer formulation can also include a buffer, such as, for example, ammonium bicarbonate, at a pH between about 7.5 and about 8, at a concentration of between about 10 mM and about 100 mM after contact with an analyte.
- a buffer such as, for example, ammonium bicarbonate
- Another stock solution of MS-compatible solubilizers can be a 5x solution comprising NDSB-201, NDSB-256, and SB-14.
- a 5x solubilizer solution can comprise NDSB-201 from about 25 mM to 650 mM, and preferably from about 50 mM to 300 mM; NDSB-256 from about 25 mM to 650 mM, and preferably from about 50 mM to 300 mM; and SB- 14 from about 0.05 mM to about ImM, and preferably from about 0.1 mM to about 0.5 mM.
- An exemplary 5x solution of a MS-compatible solubilizer of the invention comprises NDSB-201 at 125 mM, NDSB-256 at 125 mM, and SB- 14 at 1.1 mM.
- An alternative 5X formulation can be NDSB-201 at 250 mM, NDSB-256 at 250 mM, and SB-14 at 2.2 mM.
- An exemplary solubilizer can that is compatible with liquid chromatography can have a final concentration when in contact with the analyte just prior to MS analysis of, for example, from about 10 mM to about 100 mM NDSB-201, preferably from about 20 mM to about 60 mM, for example, a concentration of about 25 mM or about 50 mM; from about 10 mM to about 100 mM NDSB-256, preferably from about 20 mM to about 60 mM, for example, a concentration of about 25 mM or about 50 mM; and from about 0.1 mM to about 1 mM SB-14, preferably from about 0.1 mM to about 0.5 mM, for example, about 0.22 mM.
- a 2x solution of a MS-compatible solubilizer of the invention comprises from about 10 mM to about 1 M NDSB-201, preferably from about 20 mM to about 800 mM NDSB-201, and more preferably from about 100 mM to about 600 mM NDSB-201.
- An exemplary 2x solution of a solubilizer of the present invention comprises 500 mM NDSB-201.
- Chaotropes may be used with the matrix additives of the invention.
- Surfactants used in solubilization solutions can act synergistically with chaotropes to solubilize hydrophobic proteins, such as membrane proteins. Chaotropic agents unfold proteins, thereby exposing hydrophobic regions of the protein which can cause undesirable aggregation, precipitation, or adsorption to a solid surface. The surfactant binds to these hydrophobic domains, thus helping to keep the protein solubilized.
- urea, thiourea and guanidine hydrochloride typically at concentrations of from about 1 M to about 10 M, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 M; or at 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 M.
- the invention provides MALDI matrix additives that support and/or promote the formation of small analyte:matrix crystals in thin layers.
- the MALDI matrix additive is preferably a MS-compatible sorbent.
- MS-compatible sorbents are silicas, aluminia, germanium oxide, indium tin oxide, metal oxides, chlorides, sulfates, phosphates, carbonates and fluorides; polymer based oxides, chlorides, sulfates, carbonates, phosphates or fluorides; diatomaceous earth; graphite or activated charcoal; and titania, gold and activated gold.
- silica is preferred.
- a MS-compatible sorbent of the invention is typically, but need not be, non ⁇ volatile.
- a MS-compatible sorbent of the invention can be provided as a colloid, although other forms can be used as well.
- a MS-compatible sorbent of the invention when present at an effective amount, has one or more characteristics: (a) it increases the signal-to-noise ratio by at least about 5%; (b) it increases by at least about 5% one or more adduct cluster peaks of a molecule that forms adduct with ions; (c) it increases the stability of an analyte:matrix crystal by at least about 5%; (d) it diffracts and/or reflects the incident laser beam in a MALDI matrix comprising one or more additives to a degree sufficient to alter the fluence by at least about 1%; (e) it diffracts and/or reflects the incident laser beam in a MALDI matrix comprising one or more additives to a degree sufficient to alter the fluence at least about 10 Joules/square centimeter; and/or (f) it increases, by at least about 1%, the amount of energy that is absorbed by a MALDI matrix.
- the use of a MALDI matrix additive of the invention in MALDI-TOF MS results in a higher number of analyte molecules available for desorption and/or ionization.
- the MALDI matrix additives of the invention modify the size and/or morphology of matrix crystals and matrix:analyte crystals, such that the surface area available for laser irradiation (the "analyzable surface area") is increased.
- silica particles can be added to the matrix to reduce matrix background noise.
- a 1 :1 ratio of silica particles:matrix can be used.
- the silica particles can be provided in a variety of chemical forms including, for example, SiO 2 .
- the matrix additive can be provided in the form of a colloid, such as a colloidal solution or a resin, as a component of a diluent into which a MALDI matrix or an analyte is dissolved, or in solid form.
- a composition comprising one or more of the MS- compatible sorbents of the invention further comprises one or more ion-sequestering molecules.
- Non-limiting examples include sulfonates and zwitterionic surfactants. Ion- sequestering molecules can be introduced into the MALDI sample and/or MS matrix in colloidal form.
- a MS-compatible sorbent of the invention comprises one or more chemical compounds or compositions disclosed herein.
- Maxlon AC Other Compositions.
- a MS-compatible sorbent of the invention comprises is or comprises a resin.
- the resin can, by way of non-limiting example, be LiChrosorb®, LiChrospher®, LiChroprep®, LiChroprep® or Purospher®.
- the resin can be LiChrosorb® 5 ⁇ m, 5 ⁇ m RP8, 5 ⁇ m RP 18, LiChrosorb® 5 ⁇ m RP-Select B, LiChrosorb® 5 ⁇ m DIOL, LiChrosorb® lO ⁇ m RP 18, LiChrosorb® lO ⁇ m RP8, LiChrosorb® lO ⁇ m RP 18, LiChrosorb® 5 ⁇ m Si60 or Silica Gel 60 RP-18.
- a MS-compatible sorbent of the invention comprises is a composition comprising particles.
- the particles can comprise silica.
- the particles preferably have at least one dimension > 1 micron.
- the particles can have irregular or regular (e.g., spherical) shapes.
- a MALDI matrix additive of the invention is or comprises a MS-compatible buffer.
- the MS-compatible buffer is a morpholino- sulfonic acid.
- MS-compatible buffers include, in no particular order: 2-(n-morpholino)ethane sulfonic acid (MES); 4-(n-morpholino)butane-sulfonic acid (MOBS); 3-(n-morpholino)propane-sulfonic acid (MOPS); and 3-(n-morpholino)-2- hydroxypropanesulfonic acid (MOPSO).
- MES 2-(n-morpholino)ethane sulfonic acid
- MOBS 4-(n-morpholino)butane-sulfonic acid
- MOPS 3-(n-morpholino)propane-sulfonic acid
- MOPSO 3-(n-morpholino)-2- hydroxypropanesulfonic acid
- a MS-compatible buffer of the invention comprises one or more chemical compounds or compositions disclosed herein.
- Maxlon SA Structures of MES, MOPS and Related Compounds.
- the buffers can be used at any concentration that is compatible with MS, for example, from about ImM to about IM, or from about 10 mM to about 500 mM, or from about 20 mM to about 250 mM.
- the MS- compatible buffers of the invention improve the stability of a matrix crystal, and preferably reduce the signal-to-noise ratio in MS, particularly where multiple laser shots are used on a sample.
- the invention comprises methods of using MS-compatible reagents and/or compositions to (i) improve the solubility of an analyte by at least about 5% during one or more sample processing procedures, (ii) improves the solubility of an analyte by at least about 5% in a composition comprising a matrix, (iii) improve the stability of an analyte :matrix crystal by at least about 5%, (iv) increase the analyzable surface area of an analyte-matrix crystal by at least about 1%, (v) increase the signal-to-noise ratio by at least about 5%, and/or (vi) diminish by at least about 5% one or more adduct cluster peaks of a molecule that forms adduct with ions.
- the MS-compatible reagent is a MALDI matrix additive that is present in a matrix crystal during MS analysis.
- the MALDI matrix additive can be contacted with the sample or analyte prior to mixing the sample with the matrix, or can be provided with the matrix prior to contacting the sample or analyte with the matrix.
- the methods of the invention include without limitation processing and/or preparing samples for MS, such as but not limited to MALDI-MS and LC/MS, using one or more of the matrix additives of the invention. Included are methods of solubilizing biomolecules for MS analysis; methods of analysis using MALDI-TOF MS; methods of stabilizing analyte:matrix crystals for MALDI-MS; and methods of preparing MS target surfaces.
- the invention provides methods of using the MS-compatible reagents of the invention, such as MALDI matrix additives of the invention, in MALDI-TOF MS and other procedures for detecting, quantifying and/or studying the properties of a molecule, such as an analyte or a biomolecule.
- the invention is drawn to methods of MALDI-TOF MS analysis of a MS target molecule comprising contacting the molecule(s) with one or more of the MALDI matrix additives of the invention, and performing MALDI-MS on one or more target molecules.
- the invention provides methods and compositions for preparing a target sample for MALDI-TOF MS.
- the target sample is or comprises a crystal comprising matrix and additive molecules, or a matrix crystal comprising matrix, analyte and additive molecules.
- the matrix and analyte are combined in a single step, followed by addition of the MS matrix additive.
- the matrix and additive are combined in a single step, followed by addition of analyte.
- the matrix, analyte and additive are combined in a single step. The latter embodiment, which is or comprises a single-step matrix crystallization reaction, is preferred in some instances.
- the invention provides a method of obtaining a MALDI MS spectrum of an analyte, comprising: contacting an analyte with, in either order or in combination, a MALDI matrix; and one or more of 1) a MS-compatible solubilizer, 2) a MS-compatible sorbent, and 3) a MS-compatible buffer.
- the method further includes co-precipitating said analyte with the MALDI matrix, thus generating analyte:matrix crystals; subjecting said analyte:matrix crystals to laser irradiation, thus generating analyte ions; and detecting and quantifying the analyte ions to generate a MALDI-MS spectrum of said analyte.
- the analyte is a protein or peptide.
- the invention provides methods and compositions for producing stable matrix/analyte mixtures that can be spotted and stored on a MALDI target surface, or otherwise stored, for future analysis.
- Matrix:analyte crystals, compositions comprising such crystals, and methods of making and using such crystals are provided for in this and other aspects of the invention.
- the invention is drawn to an analyte.matrix crystal comprising one or more MS-compatible compositions.
- the MS-compatible composition can be a MS-compatible solubilizer, or a composition comprising one or more MS-compatible non- volatile MALDI additives.
- the crystals are stable, preferably under a variety of conditions.
- the method includes: contacting the analyte with a MALDI matrix and one or more of 1) a MS-compatible solubilizer, 2) a MS-compatible sorbent, and 3) a MS- compatible buffer; and co-precipitating said analyte with said MALDI matrix to generate analyte:matrix crystals for MALDI MS analysis.
- the invention is drawn to methods that comprise or involve a MS matrix additive for improving the signal-to-noise ratio during MALDI-TOF MS analysis of molecules including without limitation peptides, proteins, oligonucleotides, oligosaccharides, phospholipids, polymers, and small organic molecules. Any of these molecules can be from a biological source (“biomolecules”) or from in vitro chemical synthesis (“synthetic molecules").
- the invention provides methods and compositions for creating and/or enhancing analyzable surface areas for laser irradiation during MALDI-TOF MS. Additionally or alternatively, the extent of analyte desolvation is increased. The formation of small crystals in thin layers, which is enhanced by the additive, results in more efficient desolvation of analyte molecules, thus maximizing the number of "de-sorbable" analyte molecules.
- a matrix additive can act as an ion-exchanger that reduces or preferably eliminate the undesirable effects of ions, such as cations, including monovalent cations, and adducts of these and other ions, from MALDI spectra. More specifically, in the case of a MS-compatible sorbent, the invention provides formulations to maximize the number of deprotonated SiO 2 sites in a matrix, or in a composition used to prepare, treat or wash a crystallized matrix. These sites act as ion-exchangers for the removal of undesirable contaminants that are or result from ions, such as cations, more specifically monovalent cation contaminants.
- a desirable result of a MALDI matrix additive's positive effects on crystal size and morphology and/or capacity to act as a cation-exchanger include without limitation: (a) improvement of signal-to-noise; (b) suppression of matrix background noise; and/or (c) reduction or elimination of ion-adducts, such as monovalent cation-adducts, and/or detrimental effects resulting therefrom.
- the methods of the invention provide for the selective reduction, preferably elimination, of ion adducts, such as cation adducts, including monovalent cation adducts.
- ion adducts such as cation adducts, including monovalent cation adducts.
- an effective amount of an matrix additive of the invention involves its ability to selectively reduce and preferably eliminate monovalent cation adducts.
- Protein adducts are particularly undesirable to those studying a proteome, as it causes both the loss of molecules of interest (proteins) and production of contaminants (adducts). Both events complicate the target sample, and both can introduce inaccuracy and/or imprecision in the MS spectra.
- the adduct cluster peaks repeat at intervals of (M-I) Da, where M is the molecular mass of the cation.
- the invention provides for the reduction or elimination of the adduct cluster peaks in a MALDI-TOF MS spectrum.
- the M-I adduct cluster peak is diminished by at least about 10% upon addition of a MALDI matrix additive of the invention, preferably by at least 50% or about 50%, most preferably by 95% or about 95%, to about 100%.
- the M-I adduct cluster peak is completely diminished at 100%, but solubilizers that result in near complete elimination of the peak are also within the scope of the invention.
- the M-I adduct cluster peak is nearly completely diminished at about 90% or 90%, about 95% or 95%, about 96% or 96%, about 97% or 97%, about 98% or 98%, about 99% or 99%.
- a reference standard molecule having a known response to the solubilizing agent can be used to confirm and measure the desirable properties resulting from the presence of the solubilizer.
- One such reference standard molecule for effects of solubilizers on adducts is bradykinin, which can be tested using the compositions, methods and conditions of the invention as described in the Examples.
- a method to analyze a molecules, such as a protein, using MALDI-TOF mass spectroscopy according to the above method, wherein a monovalent cation is present in a solution that includes the molecule at the time the molecule is contacted with a MALDI matrix additive of the invention and the mass spectroscopy matrix that includes contacting the molecule with a MALDI matrix additive and a mass spectroscopy matrix, and analyzing the molecule using MALDI-TOF.
- the MALDI matrix additive can be a MS-compatible solubilizer, such as a MS-compatible detergent and/or a MS-compatible non-detergent surfactant.
- the invention provides compositions and methods for preparing a protein for MALDI-TOF analysis, the method comprising contacting a composition comprising the protein, in any order or combination, with (a) at least one MALDI matrix additive of the invention and (b) at least one enzyme, such as a protease or a protein-modifying enzyme.
- a protease or a protein-modifying enzyme.
- the enzyme is a protease.
- the invention provides compositions and methods for preparing a protein having one or more hydrophobic regions for MALDI-TOF analysis, the method comprising contacting a composition comprising said protein, in any order or combination, with (a) at least one MS-compatible solubilizer and (b) at least one enzyme, such as a protease or a protein-modifying enzyme.
- the invention provides methods of obtaining a MALDI MS spectrum of an analyte that include: 1) contacting an analyte with, in any order or combination, a MALDI matrix; and one or more of a MS-compatible solubilizer, a MS-compatible sorbent, and a MS-compatible buffer; and, at least one protease to generate one or more peptides.
- the method further includes co-precipitating the one or ore peptides with the MALDI matrix to generate analyte:matrix crystals; subjecting the analyte: matrix crystals to laser irradiation to generate peptide analyte ions, and detecting and quantifying the peptide analyte ions to generate a MALDI-MS spectrum of the one or more peptides.
- the analyte can be any type of analyte, such as, for example, a nucleic acid, a carbohydrate, or a protein.
- the invention provides methods of determining one or more amino acid sequences of a protein analyte that include: 1) contacting a protein analyte with, in any order or combination, a MALDI matrix; and one or more of a MS-compatible solubilizer, a MS- compatible sorbent, and a MS-compatible buffer; and, at least one protease to generate one or more peptides.
- the method further includes co-precipitating the one or ore peptides with the MALDI matrix to generate analyte :matrix crystals; subjecting the analyte.matrix crystals to laser irradiation to generate peptide analyte ions, and detecting and quantifying the peptide analyte ions to generate a MALDI-MS spectrum of the one or more peptides, and using the MALDI-MS spectrum to determine the sequences of the one or more peptides, where the sequences of the peptides are sequences of the protein analyte.
- the invention provides methods of determining an amino acid sequence of a protein analyte that binds to a ligand that include: 1) contacting a protein analyte with, in any order or combination, a MALDI matrix; and one or more of a MS-compatible solubilizer, a MS-compatible sorbent, and a MS-compatible buffer; and, 2) contacting a second sample comprising said protein analyte with, in any order or combination, a MALDI matrix; one or more of a MS-compatible solubilizer, a MS-compatible sorbent, and a MS-compatible buffer; and a ligand of the protein analyte.
- the method further includes 3) independently contacting the first and second samples with a protease, to generate a first set of one or more peptides and a second set of one or more peptides and 4) independently co-precipitating the rist and second set of peptides with the MALDI matrix to generate first and second analyte:matrix crystals.
- the method further includes 5) subjecting the analyte:matrix crystals independently to laser irradiation, thus generating first and second sets of peptide analyte ions and 6) detecting and quantifying the peptide analyte ions, to generate a MALDI-MS spectrum of the first and second sets of peptides; and using the MALDI-MS spectrum to determine the amino acid sequences of the first and second sets of peptides, in which an amino acid sequence depleted in the sequences of the second set set relative to the first set is an amino acid sequence of a protein analyte that binds the ligand.
- the invention provides methods of preparing a hydrophobic molecule for MS analysis, the methods comprising contacting a composition comprising said hydrophobic molecule with at least one MS-compatible solubilizer.
- the hydrophobic molecule for example, can be a membrane protein.
- membrane proteins can be analyzed using mass spectrometry for chemically modified sites, ligand binding sites, and component peptide sequences.
- the present invention expands the useful applications of MS to hydrophobic molecules including membrane proteins.
- composition can comprise one or more enzymes, one or more chaotropes, and/or one or more co-additives.
- Co-additives include without limitation phospholipids, fatty acids, cholesterol, steroid compounds and organic solvents. Such co- additives help to separate hydrophobic molecules from other molecules, including molecular complexes or other hydrophobic molecules in a sample.
- co-additives can be used to help separate the protein of interest from a molecular complex, or to help displace a membrane protein of interest from membranes.
- the invention provides compositions and methods of preparing a sample comprising a protein, such methods comprising (a) subjecting a sample comprising the protein to a process that at least partially separates the protein from other molecules in the sample, to generate a partially purified protein, and (b) contacting a composition comprising the partially purified protein with MALDI matrix additive of the invention, thereby generating a sample comprising the protein suitable for MALDI-TOF analysis.
- An enzyme such as a protease, and/or a matrix suitable for MALDI-TOF may also be added at (b) or some other point in sample preparation.
- a preferred MALDI matrix additive is a MS-compatible solubilizer.
- the invention provides methods of obtaining a MS-MALDI spectrum of a protein analyte, methods of determining one or more amino acid sequences of a protein analyte, methods for identifying an amino acid sequence of a protein analyte that binds to a ligand, methods for identifying an amino acid sequence of a protein analyte that is chemically modified by a protein-modifiying enzyme.
- Protein-modif ⁇ ying enzymes include without limitation kinases, phosphatases,glycosylases, deglycosylases, and combinations and complexes thereof.
- the invention provides compositions and methods for preparing a protein for MALDI-TOF analysis, the method comprising contacting a composition comprising the protein, in any order or combination, with (a) at least one MALDI matrix additive of the invention and (b) at least one enzyme, such as a protease or a protein-modifying enzyme.
- a protease or a protein-modifying enzyme.
- the enzyme is a protease.
- the invention provides compositions and methods for preparing a protein having one or more hydrophobic regions for MALDI-TOF analysis, the method comprising contacting a composition comprising said protein, in any order or combination, with (a) at least one MS-compatible solubilizer and (b) at least one enzyme, such as a protease or a protein-modifying enzyme.
- the invention provides compositions and methods for identifying a region on a molecule that binds to a region on a ligand, comprising contacting the molecule and/or the ligand with at least one MALDI matrix additive of the invention, thereby generating a sample suitable for MALDI-TOF analysis, and subjecting the sample to MALDI-TOF analysis.
- the molecule and/or the ligand can be hydrophobic, or comprise at least one region that is hydrophobic, in which case a preferred MALDI matrix additive is a MS-compatible solubilizer of the invention.
- the invention provides compositions and methods for identifying a protein that binds to a ligand, the method comprising (a) contacting, in any order or combination, (i) a sample comprising one or more proteins, (ii) the ligand, (iii) one or more cross-linkers, (iv) a MALDI matrix additive of the invention, and (v) a protease; in order to generate cross-linked peptides, which are cross-linked to the ligand or some portion thereof, and determining the amino acid sequences of the cross-linked peptides by MALDI- MS analysis.
- the amino acid sequence of the cross-linked peptides comprise all or part of a region on a protein that binds to said ligand.
- the matrix additive can be any disclosed herein, such as for example, a MS-compatible buffer, sorbent, or solubilizer.
- the invention provides compositions and methods for identifying a protein or region thereof that is chemically modified, said method comprising: (a) contacting, in any order or combination, (i) the protein, (ii) an enzyme that modifies proteins, (iii) a MALDI matrix additive of the invention, and (iv) a protease, in order to generate chemically modified peptides.
- the amino acid sequences of the chemically modified peptides are determined by MALDI-TOF analysis; these amino acid sequences comprise all or part of a region on a protein that is chemically modified by the enzyme.
- the matrix additive can be any disclosed herein, such as for example, a MS-compatible buffer, sorbent, or solubilizer.
- the invention also provides compositions and methods for extending sequence coverage in peptide-mass fingerprinting, comprising contacting the peptide with a MALDl matrix additive of the invention and performing MALDI-MS on the peptide.
- the sequence coverage of the peptide is greater than that of the peptide analyzed by MALDI-MS in the absence of the matrix additive.
- the invention provides compositions and methods for inhibiting the formation of protein:ion adducts in a protein, comprising contacting the protein with a MALDI matrix additive of the invention and performing MALDI-MS on the peptide.
- the invention also provides compositions and methods for evaluating uncharacterized compounds and compositions for their potential as matrix additives of the invention (e.g., MS-compatible solubilizers, MS-compatible sorbents and/or MS-compatible buffers).
- matrix additives of the invention e.g., MS-compatible solubilizers, MS-compatible sorbents and/or MS-compatible buffers.
- Matrix-assisted laser desorption time-of- flight mass spectrometry typically involves several processes, e.g., matrix formation (co-crystallization), desorption, desolvation and ionization. Matrix formation involves mixing analyte molecules with an excess of matrix molecules and co-crystallizing the two.
- the matrix is an acidic aromatic, e.g., sinapinic acid (SA) or alpha-cyano-4-hydroxycinnamic acid (CHCA).
- SA sinapinic acid
- CHCA alpha-cyano-4-hydroxycinnamic acid
- the matrix molecules are selected to be capable of absorbing light at wavelengths compatible with an emitting laser.
- Analyte molecules are complexed with and/or surrounded by matrix molecules as they leave the surface. After a short distance the analyte molecules begin to desolvate and separate from the matrix, allowing them to be ionized.
- Ionization can occur as analyte molecules are released and/or after desolvation.
- the analyte molecules are ionized from matrix clusters in a series of collision events. Ions are accelerated into a time-of- flight tube where they are separated by their momentum.
- the selection of matrix varies depending on the nature of the analyte being analyzed. Generally, however, effective MALDI matrices share common physical and chemical characteristics. (1) the matrix must effectively associate with the analyte to break-up intermolecular aggregation of analyte molecules, and to assure even distribution of the analyte within the sample spot; (2) the matrix must be stable under vacuum conditions; (3) the matrix must absorb at wavelengths compatible with the emitting laser. Moreover, the solubility properties of the analyte must match that of the matrix molecule, so that they are soluble within the same volatile solvent. For any given analyte of interest, identification of an optimal matrix, ratio of matrix:analyte, analyte concentration, solvent selection, spotting method, sample preparation conditions and instrument conditions is desirable for best results involving that specific analyte.
- conditions vary from analyte to analyte. That is, conditions for MALDI for each specific analyte vary and must be optimized in terms of, e.g., choice of matrix, treatment of the matrix: analyte during MALDI, etc.
- matrix additives might be used, or the matrix or analyte might be pretreated, etc. All these parameters are typically determined empirically. Preparation of samples for MALDI is presently as much art as science.
- matrix and “matrix molecules” refer to the material with which a biomolecule can be combined for MALDI mass spectrometric analysis. Any substance that can absorb light at the laser's wavelength (300-400 nm) and is crystal-forming can be used. Any matrix material, such as solid acids, including 3-hydroxypicolinic acid and alpha-cyano-4-hydroxycinnamic acid (a.k.a. gentisic acid, CHCA, 4-HCCA), and liquid matrices, such as glycerol, known to those of skill in the art for MALDI-TOF MS analyses is contemplated. Materials useful for matrix formulation include without limitation 4-HCCA (a.k.a.
- CHCA sinapinic acid
- DHBA 2,5-dihydroxybenzoic acid
- HPA 3-hydroxy-picolinic acid
- nor-harmane is prepared as a 10 mg/ml solution in 50% acetonitrile/50% water for aqueous soluble molecules, tetrahydrofuran for polymers and chloroform for lipids.
- SA is recommended for preparations of intact hydrophobic proteins and 4-HCCA is recommended for enzymatic digestion of proteins.
- Sinapinic acid which is mostly used for analysis of intact proteins, has a fragile crystal structure that becomes ablated during prolonged exposure to the MALDI laser. Thus, this fragility precludes enhancement of the signal-to-noise of low abundance proteins through longer acquisitions. While protein identification and characterization relies to a great extent on the study of a set of accurate mass measurements derived from proteolytic digests, exact mass measurement of intact proteins still play an important role, especially in the study of post-translational modifications. Sinapinic acid (SA) is the matrix of choice for large proteins.
- Alpha-cyano-4-hydroxycinnamic acid which is mostly used for analysis of peptides, requires specific spotting techniques and the use of specific solvents to generate a homogeneous distribution of small crystals, which in turn produce the highest quality spectra.
- Alpha-cyano-4-hydroxycinnamic acid is also referred to herein as "alpha c", “ ⁇ C”, “alpha- cyano”, “ ⁇ -cyano”, “CHCA” and "HCCA”.
- Target analyte matrix crystals for MALDI are typically prepared by mixing and co-precipitating/co-crystallizing a matrix and an analyte, wherein the matrix, often an acidic aromatic matrix, is a molecule capable of absorbing light at wavelengths compatible with an emitting laser.
- the matrix/analyte mixture is subject to laser, the matrix molecules absorb the laser energy and are desorbed from the target surface by vaporization, forcing the co-desorption of the analyte molecules with which they are co-crystallized.
- the analyte molecules undergo ionization as they are ionized in a series of collision events, and accelerated into a time-of-flight tube where they are separated by their momentum.
- a sample is prepared, mixed with a suitable matrix, and deposited on the MALDI target to form dry mixed crystals and, subsequently, placed in the source chamber of the mass spectrometer.
- protein identification by this technique has the advantage of short measuring time (typically, a few minutes) and negligible sample consumption (less than 1 pmol) together with additional information on microheterogeneity (e.g., glycosylation) and presence of byproducts.
- the "dried-droplet" method of sample preparation is relatively simple. A saturated solution of matrix material is mixed with protein to a final concentration of 1-10 mM. A droplet (0.5-2 microliters) of the resulting mixture is placed on the mass spectrometer's sample stage. The droplet is dried at room temperature and, when the liquid has completely evaporated, the sample may be loaded into the mass spectrometer. Dried droplets are relatively stable and can be kept out of direct light and/or in vacuum for days.
- the "slow crystallization" method may improve detection, particularly of larger proteins (Cohen et al., Anal Chem. 68:31, 1996; Botting, Rapid Commun Mass Spectrom. 14:2030, 2000).
- the protein sample is thoroughly mixed with the matrix solution and Iety stand for a few hours. Large crystals, formed on the walls of the micro fuge tube, are washed with water, scraped off, and applied to the MALDI target.
- Polycrystalline thin films can be used in MALDI-TOF MS sample preparation. This method of sample preparation produces a uniform layer of very small crystals on the mass spectrometer's sample stage that are mechanically well adhered to the substrate. The crystals can be thoroughly washed without removing them from the surface.
- Li et al. (Dai et al., Anal Chem. 71 :1087, 1999; Zhang et al., Anal Chem. 73:2968, 2001) developed a 2-layer sample preparation technique. This approach involves formation of a microcrystalline layer of matrix using fast solvent evaporation, followed by a deposition of a mixture of matrix and sample on top of the microcrystlline layer. The film grows rapidly, so it is not necessary to wait until the droplet is dry before washing the film, reducing effects caused by increasing contaminant concentrations as the droplet dries.
- the invention is drawn to MALDI matrix additives or, more simply, matrix additives.
- matrix additives are included in matrices for the purpose of enhancing or adding a desirable characteristic to the matrix and/or matrix :analyte co- crystal.
- a matrix additive of the invention including without limitation a non-volatile matrix additive, can be mixed with MALDI matrix to initiate, promote, accelerate and/or support the formation of small crystals in thin layers in a single step, thus providing a large analyzable surface area for laser irradiation.
- the formation of small crystals in thin layers aided by the additive produce the most efficient desolvation of analyte molecules, thus maximizing the number of desorbable analyte molecules, which results in better ionization of the analyte molecules.
- a matrix additive of the invention acts as an ion-exchanger, resulting in removal of monovalent cation contaminants.
- a MALDI matrix additive of the invention results in improved signal-to-noise, reduction of monovalent cation-adducts, and suppressed matrix background noise.
- MALDI SA Matrix Additive As it is typically but not exclusively used in SA-based matrices, one type of formulation is referred to herein as a MALDI SA Matrix Additive.
- a specific but non- limiting of a MALDI SA Matrix Additive is Maxlon SA, which is described in more detail herein. Maxlon SA utilizes a novel diluent formulation that attenuates laser-induced damage of SA crystals and thus allows an increased number of MALDI acquisitions to be made and summed, thus enhancing the overall MALDI-MS spectral quality.
- MALDI CHCA Matrix Additive As it is typically but not exclusively used in CHCA-based matrices, one type of formulation is referred to herein as a MALDI CHCA Matrix Additive.
- a specific but non- limiting of a MALDI CHCA Matrix Additive is Maxlon AC, which is described in more detail herein.
- Maxlon AC includes one novel diluent based on a solubilizer (the zwitterionic surfactant NDSB) and silica resin as a MALDI matrix additive.
- Silica is an efficient matrix crystal morphology modulator that enhances spectral quality even under high salt conditions.
- the silica resin additive promotes the formation of thin layers of small CHCA crystals thus reducing the matrix background, enhancing ionization of low-abundance species, and eliminating the suppressive effects of salt contamination.
- the invention provides methods and compositions for analyzing proteins.
- the invention can be used to detect, quantify, identify, characterize a protein.
- Some specific examples of analysis that the invention is suited for include, but are not limited to: (1) amino acid sequence determination and peptide mass fingerprinting (PMF), useful for identifying a gene encoding a protein of interest; (2) determination of sites of chemical modifications of proteins; (3) identification of ligand binding proteins and domains, useful in studies of interactions of protein with other proteins and molecules, and pharmacology/drug discovery.
- PMF amino acid sequence determination and peptide mass fingerprinting
- the target molecule to be analyzed using mass spectrometry can be a hydrophilic molecule, e.g., a hydrophilic protein, such as a soluble protein or a small synthetic molecule, including oligonucleotides or peptides.
- the target molecule can be a hydrophobic molecule, e.g., a hydrophobic protein, such as a membrane protein.
- Hydrophobic proteins of interest include ion channels or a transporters, particularly ligand-gated ion channels, such as a serotonin receptor, a gamma-aminobutyric acid receptor, a glycine receptor, a glutamate- gated chloride channel, a glutamate receptor, an ATP-gated channel, or an NMDA receptor.
- ligand-gated ion channels such as a serotonin receptor, a gamma-aminobutyric acid receptor, a glycine receptor, a glutamate- gated chloride channel, a glutamate receptor, an ATP-gated channel, or an NMDA receptor.
- Other hydrophobic proteins of interest have at least one transmembrane domain and/or homology to the nicotinic acetylcholine receptor of Torpedo californica. Peptide Mass Fingerprinting CPMF)
- a peptide mass fingerprint is a compilation of the molecular weights of peptides generated by a specific protease. More recently, the ability to determine all or part of the amino acid sequence of the protein fragments. The sequence information, as well as data relating to the molecular weights of the parent protein prior to protease treatment and the subsequent proteolytic fragments is used to search genome databases for any similarly sized protein with identical or similar amino acid sequences and/or peptide mass maps
- Proteases useful in PMF and amino acid sequencing include without limitation trypsin, chymotrypsin, elastase, Endoproteinase Arg-C, Endoproteinase Asp-N, Endoproteinase GIu-C, Endoproteinase Lys-C, Aminopeptidase M, Carboxypeptidase-Y and pronase.
- protease cleavage reactions see Sweeney, P. and Walker, J.M. Chapters 14-18 in: Enzymes of Molecular Biology, Methods in Molecular Biology 16, M. M. Burrell (ed.), Humana Press, Totowa, NJ (1993).
- Chemical proteolysis e.g., Edman degration
- MS-Digest allows for the in silico digestion of a protein sequence with a variety of proteolytic agents including trypsin, chymotrypsin, V8 protease, Lys-C, Arg-C, Asp-N, and CNBr.
- the program calculates the expected mass of fragments from these virtual digestions and allows the effects of protein modifications such as N-terminal acetylation, oxidation, and phosphorylation to be considered. Modifications of Proteins
- compositions and methods described herein can be used to study chemical and enzymatic modification and, in particular, to identify the sites (amino acid sequences) where the modifications occur.
- Post-translational modifications can be studied in this fashion.
- proteins that are modified in signaling, and other cellular pathways, including apoptosis can be studied.
- proteins to which a phosphate group is added (by kinases) or removed (by phosphatases) often occur in cellular pathways. Examples of such studies include without limitation the following.
- Protein-modifying enzymes that can be used in the invention include without limitation kinases, phosphatases, glycosylases and deglycosylases. See, e.g., Jaquinod et al., Biol Chem 380:1307-1314 (1999); Kuster et al., Curr Opin Struct Biol. 8:393-400 (1998); Yan et al., Biochem Biophys Res Commun 259:271-282 (1999); and Nilsson, MoI Biotechnol 2:243-280 (1994).
- MALDI-TOF MS can also be used to obtain information about quaternary structures, such as mapping of protein-protein contacts or ligand binding sites.
- MALDI-TOF MS has been used to study the binding of alpha-neurotoxin to the nicotinic acetylcholine receptor and substance P to the neurokinin- 1 tachykinin receptor, and the mapping of the agonist binding site of the cholecystokinin B receptor (Macho Id et al., Proc Natl Acad Sci USA 92:7282, 1995; Girault et al., Eur J Biochem 240:215, 1996; Anders et al., Biochemistry 38:6043, 1999).
- Detectably-labeled ligands such as known agonists, antagonists, receptor ligands, and derivatives thereof, are covalently linked to the binding site in a protein of interest in cross-linking reactions. Subsequent proteolysis and MALDI-TOF MS analysis result in PMF maps with additional peaks, indicating the cross-linking site on the protein.
- Photoaffmity labeling represents one type of cross-linking strategy that can be used to identify and characterize those regions of a protein in which an interaction with low- molecular-mass ligands takes place.
- photoaffinity probes include without limitation [3H]4-Benzoylbenzoylcholine (Wang et al., J. Biol. Chem., 275:28666, 2000), [3H]nicotine (Middleton et al., Biochemistry 30:6987, 1991) and p-(N,N- dimethyl)aminobenzenediazonium fluoroborate (Galzi et al., J. Biol. Chem. 256:10430, 1990).
- Ligand-binding regions of glycoprotein P have also been studied using photoaffinity labeling and MALDI-TOF MS (Ecker et al., MoI Pharmacol. 61 :637, 2002).
- the invention provides compositions and methods for studying, for example, hydrophobic proteins, including without limitation membrane proteins.
- Proteins that span a biological membrane are said to have one or more transmembrane domains (TMDs).
- TMDs transmembrane domains
- Membrane proteins may represent as much as one half of the total diversity of some proteomes, and play many roles in fundamental biological processes such as cell-cell interactions, cell signaling, protein trafficking, ion and solute transport and intracellular compartmentalization. Many pharmacological targets are membrane proteins.Membrane and other hydrophobic proteins are notoriously difficult to study with conventional methods.
- Membrane protein can be from any biological source membrane, including without limit cellular membranes, viral envelopes (Kim et al., Anal Chem.
- membranes from an organelle such as a nucleus, a nucleolus, a mitochondrion, a chloroplast, and the endoplasmic reticulum.
- organelle such as a nucleus, a nucleolus, a mitochondrion, a chloroplast, and the endoplasmic reticulum.
- mitochondria and chloroplasts both the inner and outer membranes, and the intermembrane space, can be sources of membrane and other hydrophobic proteins.
- the invention can be applied to any membrane protein, including but not limited to the following exemplary receptors and membrane proteins.
- the proteins include but are not limited to receptors (e.g., G-protein coupled receptors, or GPCRs, sphingolipid receptors, neurotransmitter receptors, sensory receptors, growth factor receptors, hormone receptors, chemokine receptors, cytokine receptors, immunological receptors, and compliment receptors, FC receptors), channels (e.g., potassium channels, sodium channels, calcium channels.), pores (e.g., nuclear pore proteins, water channels), ion and other pumps (e.g., calcium pumps, proton pumps), exchangers (e.g., sodium/potassium exchangers, sodium/hydrogen exchangers, potassium/hydrogen exchangers), electron transport proteins (e.g., cytochrome oxidase), enzymes and kinases (e.g., protein kinases, ATPases, GTPases, phosphatases, proteases (
- LGICs Ligand-Gated Ion Channels
- LGICs include a serotonin (5-hydroxytryptamine or 5-HT) receptor (e.g., 5-HT3A and 5- HT3B); a gamma-aminobutyric acid receptor; a glycine receptor; a glutamate-gated chloride channel; a glutamate receptor; an ATP-gated channel; and an NMDA receptor.
- 5-HT 5-hydroxytryptamine
- 5-HT3A and 5- HT3B a gamma-aminobutyric acid receptor
- a glycine receptor a glutamate-gated chloride channel
- glutamate receptor an ATP-gated channel
- NMDA receptor an NMDA receptor
- Cys-loop refers to the presence of a pair of disulphide-bonded cysteines near the N-terminal of the protein.
- Cys loop LGICs is assembled from a pool of more than 40 homologous subunits. These subunits have been classified into four families representing channels that are gated by acetylcholine, serotonin, gamma-aminobutyric acid, or glycine.
- the muscle-type nicotinic acetylcholine (ACh) receptor (mnAChR) is an exemplary cys-loop protein.
- the basic structural features of mnAChRs (four membrane- spanning segments, ligand-binding sites at subunit interfaces and a pore formed by M2) are thought to be preserved in the other members of the cys-loop superfamily.
- Two members of the cys-loop superfamily, gamma-aminobutyric acid type A receptors (GABAAR) and glycine receptors (GIyR) are permeable to anions rather than cations.
- Neuronal nicotinic ACh receptors include without limitation nAchR.
- the ionotropic glutamate receptor (GIuR) superfamily consists of three families, all of which are activated in vivo by L-glutamate. The three families are distinguished by their affinity for the synthetic agonists -amino-5-methyl-3-hydroxy-4- isoxazole propionic acid (AMPA), N-methyl-D-aspartame (NMDA) and kainate.
- AMPA -amino-5-methyl-3-hydroxy-4- isoxazole propionic acid
- NMDA N-methyl-D-aspartame
- kainate ionotropic, purinergic receptor (P2X) ATP-activated superfamily of LGICs exhibit two membrane-spanning regions and no pore-loops. All of the receptors are about equally permeable to Na and K and also have significant Ca permeability.
- Volatile anaesthetics and alcohols e.g., soflurane and butanolanaesthetics (ether, cyclopropane, butane)
- mnAChRs McLarnon JG, Pennefather P, Quastel DMJ. Mechanisms of nicotinic channel blockade by anesthetics. In: Roth SH, Miller KW, eds. Molecular and Cellular Mechanisms of Anesthetics. New York: Plenum Press, 1986; 155-164).
- the ligand-binding domain of LGICs has been imaged at 1.9 A resolution to reveal a clamshell-shaped shape having two lobes surrounding a large binding cleft.
- An Ach-binding protein isolated from snail glial cells has an amino acid sequence that resembles the extracellular portion of members of the cys-loop superfamily, and like the mammalian channels, it forms a pentamer.
- the methods of the invention can be applied to known proteins, domains and/or amino acid sequences, or to uncharacterized proteins, domains and/or amino acid sequences that are substantially identical and/or have homology to each other.
- nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% or about 60% identity, optionally 65% or about 65%, 70% or about 70%, 75% or about 75%, 80% or about 80%, 85% or about 85%, 90% or about 90%, or 95% or about 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using sequence comparison algorithms or by manual alignment and visual inspection.
- sequences are then said to be “substantially identical.”
- identity exists over a comparison window or designated region that is at least 25 or about 25 nucleotides (nt) or amino acids (aa) in length, more preferably over a region that is from 25 or about 25 to 75 or about 75 nt or aa in length, even more preferably from about 75 or 75 to 150 or about 150, or more, nt or aa in length.
- polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence it is intended that the amino acid sequence of the polypeptide is identical to the reference sequence, except that the polypeptide sequence may include up to five amino acid alterations, including deletions and insertions, per each 100 amino acids of the reference amino acid sequence.
- the polypeptide sequence may include up to five amino acid alterations, including deletions and insertions, per each 100 amino acids of the reference amino acid sequence.
- up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, and/or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
- a "comparison window" includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 or about 20 to 600 or about 600, usually from 50 or about 50 to 200 or about 200, more usually 100 or about 100 to 150 or about 150, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
- Methods of alignment of sequences for comparison include the following. Alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.
- the overall homology of a protein to another protein, or of a domain or motif to another can be 50% or about 50%, 60% or about 60%, 70% or about 70%, 75% or about 75%, 80% or about 80%, 85% or about 85%, 90% or about 90%, 95% or about 95% or 99% or about 99%.
- the percent homology between two sequences is determined using sequence analysis software. Such software matches similar sequences by assigning degrees of homology to various insertions, deletions, substitutions, and other modifications.
- Exemplary software include: The Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, WI; (Devereux et al., Nucleic Acids Res. 12:387, 1984); The algorithm of E. Myers and W.
- Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389, 1997).
- the default parameters of the respective programs e.g., XBLAST and NBLAST
- a number of sequence databases can be searched for homologous molecules, including, for example, the GenBank database (National Center for Biotechnology Information, Bethesda), EMBL data library (European Bioinformatics Institute, Cambridge, UK), the Protein Sequence Database and PIR-International, and SWISS-PROT.
- GenBank database National Center for Biotechnology Information, Bethesda
- EMBL data library European Bioinformatics Institute, Cambridge, UK
- PIR-International European Bioinformatics Institute
- SWISS-PROT SWISS-PROT.
- kits that include one or more compositions of the invention.
- a kit can comprise containers consisting of one, two or more compounds or mixtures thereof in either a solution or in powdered (dessicated or lyophilized) form. Such compounds and mixtures include the MS-compatible solubilizing formulations described herein, and/or the non-volatile MS matrix additives, and optionally several dilutions thereof.
- a kit can optionally further comprise one or more other kit components, including but not limited to one or more chaotropes, optionally mixed in an optimized proportion; one or more MALDI matrices; one or more buffers; one or more standard or control proteins and/or one or more MS calibrants.
- Containers are typically sealed and can be, e.g., a packet, a bag, a vial, a tube, a blister pack, a microtiter plate or any other suitable container.
- kits hi one embodiment of this aspect of the invention, a kit of the invention comprises one or more MS-compatible solubilizers.
- the MS-compatible solubilizer can be one described herein.
- the MS-compatible solubilizer comprises a compound selected from the group consisting of ASB-C80, Octyl-beta-D-l-thioglucopyranoside, n-Dodecanoylsucrose, SB14 and a non- detergent sulfobetaine.
- the MS-compatible solubilizerin a kit is typically provided in the form of a concentrated stock solution, e.g., a 1.5x, 2x, 3x, 4x, 5x, 1Ox, 15x, 25x, 50x orlOOx stock solution.
- the kit further comprises one or more matrix compositions.
- matrix compositions include sinapinic acid and alpha-cyano-4- hydroxycinnamic acid.
- the kit further comprises one or more matrix solvents.
- matrix solvents inlcude 0.1% trifluoroacetic acid and 100% acetonitrile.
- the kit further comprises one or more chaotropes.
- chaotropes include urea, thiourea and guanidine chloride.
- the kit further comprises one or more enzymes, such as a protease.
- proteases include TEV protease, trypsin, chymotrypsin, elastase, Endoproteinase Arg-C, Endoproteinase Asp-N, Endoproteinase GIu-C, Endoproteinase Lys- C, Aminopeptidase M, Carboxypeptidase-Y and pronase.
- the kit further comprises one or more buffers; one or more cross- linkers; one or more standards, controls or calibrants; and a product manual that describes storage conditions and one or more experimental protocols.
- experimental protocols inlcude a protocol for direct analysis and calibration of intact hydrophobic proteins, a buffer exchange protocol, and a trypsin digestion protocol.
- a kit of the invention comprises (a) a container comprising a solution of ASB-C80, Octyl-beta-D-1-thioglucopyranoside, n-Dodecanoylsucrose and SB 14; (b) a container comprising NDSB-201 ; and, optionally, one or more of: (c) a container comprising one or more molecular weight standards; (d) a container comprising sinapinic acid; (e) a container comprising alpha-cyano-4-hydroxycinnamic acid; (f) a container comprising trifluoroacetic acid; and (g) a container comprising acetonitrile.
- a kit of the invention comprises (a) a container comprising 10 ml of a solution of ASB-C80 at 125 mM, Octyl-beta-D-l-thioglucopyranoside at 50 raM, n- Dodecanoylsucrose at 3.8 mM, and SB 14 at 1 mM; (b) a container comprising 25 ml of 500 mM NDSB-201; and, optionally, one or more of: c) a container comprising 25 ⁇ L of 90 kDa InvitroMass protein standard; (d) a container comprising 20 mg of sinapinic acid; (e) a container comprising 20 mg of alpha-cyano-4-hydroxycinnamic acid; (f) a container comprising 20 ml of 0.1% trifluoroacetic acid; and (g) a container comprising 1 ml of 100% acetonitrile.
- kits can include (a) a container comprising a solution of NDSB 201, NDSB 256, and SB 14; and (b) a container comprising a protein standard.
- a kit of the invention comprises (a) a container comprising 5 ml of a solubilizer solution of 125 mM NDSB 201, 125 mM NDSB 256, and 1.1 mM SB14 in 125 mM Ammonium bicarbonate, pH 7.8; and (b) a container comprising 50 microliters of a standard tryptic digest of BSA in IX solubilizer.
- kit Instructions may also be included in a kit. Typically, sufficient documentation will be included to describe the application of this kit's components to the direct analysis and calibration of intact hydrophobic proteins; general recommendations for contaminants, particularly SDS, Triton X, CHAPS, and HEPES; and a description of performance and analysis of tryptic digests supported by the solubilizers.
- a kit may also comprise one or more solid supports, which can optionally be coated with one or more one or more MS-compatible compositions of the invention.
- Such solid supports include without limitation beads, porous beads, crushed particles, membranes, tubing and planar surfaces (e.g., plates). Descriptions of representative kits of the invention are given in the Examples herein.
- EXAMPLE 1 ANALYSIS OF DETERGENTS AND OTHER
- This Example illustrates testing of detergents and other surfactants to identify solubilizer formulations that can be used for methods of the present invention that involve MALDI-TOF-MS analysis.
- the formulations include surfactant molecules that have been independently tested for suppression effects on the ionization of peptides and intact proteins by MALDI.
- a MALDI-MS compatible surfactant blend formulation was devised by separately assaying the effect of individual components on the ionization efficiency of a peptide mixture.
- the performance of BLEND I in MALDI-TOF MS was tested using beta- galactosidase (b-gal) and BSA.
- Bovine serum albumin (BSA) a commonly utilized test protein, was used as an exemplary intact protein, and a tryptic digest of b-galactosidase (t-b- gal) was used as an exemplary peptide mixture.
- BSA bovine serum albumin
- t-b- gal tryptic digest of b-galactosidase
- b-gal is a commonly utilized test protein; moreover, the b-gal tryptic fragments represent a range of solubility from hydrophilic to hydrophobic.
- MALDI-MS analysis of t-beta-gal in a solution containing the test surfactant was carried out separately for each surfactant.
- Table 4 lists the various solutions of t-beta-gal and detergent that were examined. Each detergent was tested at a concentration near its CMC, the exception being ASB-C80, for which there is no CMC data. Samples were run in parallel with an equivalent control sample of t-beta-gal containing no surfactant.
- MALDI-MS analysis revealed that most components (with the exception of SB 10) do not significantly suppress ionization, since total ion counts were similar for the test samples and the control sample.
- n-Dodecanoylsucrose suppressed t-beta-gal peptides the least, and SB 10 suppressed the most.
- n- Dodecanoylsucrose matched 100% of the mass-ions identified in the t- beta-gal sample without surfactant (MASCOT® score 133, 14% sequence coverage).
- ASB-C80 (a.k.a. ASB-C8F) is 4-n-Octylbenzoylamido-propyl-dimethylammonio sulfobetaine.
- **SB10 (a.k.a. sulfobetaine 10) is n-Decyl-N,N-dimethyl-c-ammonio-l-po ⁇ anesulfonate.
- ***SB14 (a.k.a. sulfobetaine 14) is n-Tetradecyl-N,N-dimethy-3-ammonio-l- propanesulfonate.
- Table 4 also lists the percentage of match for each individual surfactant for the m/z 1199-3180 data range.
- the MALDI-MS data was analyzed further to identify the t-beta- gal fragment that corresponded to individual mass-ions that had been selectively suppressed in the presence of surfactant.
- 8 contained one or more tryptophan residues
- 10 contained two or more aromatic residues. The mechanism of this suppression is not known and does not appear to be surfactant specific, although the selective suppression effect was most pronounced with SB 14.
- Calbiochem® is a brand of EMD Biosciences, Inc. (San Diego, CA).
- Fluka is a brand of Sigma-Aldrich Corp. (St. Louis, MO).
- BLEND I does not interfere with ionization and sensitivity of the MALDI-MS analysis of peptides and proteins.
- the surfactant blend must be an efficient solubilization agent.
- the performance characteristics of BLEND I were tested as follows. [00308] Drop-dialysis on Cytochrome P450 1A2 (Jnvitrogen/PanVera) was carried out in order to exchange the 20% glycerol included in the stock storage buffer for BLEND I.
- Cytochrome P450 was selected as the test protein because it contains a transmembrane segment within the first 30 N-terminal residues and thus requires a surfactant to be soluble in an aqueous solution.
- Drop-dialysis was performed using a 25 mm filter-membrane (Millipore) placed on top of an inverted 15 mL conical tube cap containing 3 ml of 0.05X BLEND I.
- Three (3) ⁇ L of Cytochrome P450 (1.7 ⁇ g/ ⁇ L) was mixed with 3 ⁇ L of IX BLEND I and incubated at RT for 10 min. The 6 ⁇ L drop was then placed on the membrane and dialysed for 2 hrs, followed by a bottom buffer change and an additional 2 hrs.
- detergent monomers, salts and glycerol are allowed to freely equilibrate between the drop and the bottom buffer.
- Detergent micelles and protein are retained at the drop due to the ⁇ 2,500 Da molecular weight cut-off of the filter.
- the sample contains 8 x 10 ⁇ 5 % glycerol. It should be noted that the increase from the relatively high concentration of glycerol caused the volume of the drop to increase three to five fold (swelling often occurs at concentrations of glycerol >20%).
- the MALDI-MS spectra of Cytochrome P450 prior to and after dialysis show that 20% glycerol suppresses the ionization of Cytochrome P450, while exchange for BLEND I relieves this suppression.
- the negative control water
- Detergent exchange may be performed in the presence of the 90 kDa InvitroMass calibrant. Besides providing an internal mass standard (alternatively the standard can be spiked into the analyte solution immediately prior to MALDI-MS analysis), the inclusion of the standard is a positive control for testing the possible residual presence of interfering detergent.
- EXAMPLE 3 ENHANCED SEQUENCE COVERAGE OF THE PEPTIDE
- EXAMPLE 4 ENHANCED SEQUENCE COVERAGE OF THE
- nAChR nicotinic acetylcholine receptor
- Figure 1 shows the results of SDS-PAGE of purified T. californica nAChR.
- the electrophoretic migration pattern of the individual subunits (alpha, beta, gamma and delta) that assemble into the native, pharmacologically active nAChR is shown in relation to molecular weight markers. The inset to the right more specifically identifies each subunit of the nAChR.
- Bands were excised manually and destained in 50% acetonitrile/ 25 mM ammonium bicarbonate (ABC) at pH 7.8 (ABC-7.8) or 8.0 (ABC-8.0) until blue color was no longer visible. Bands were dehydrated with 200 ⁇ L of 100% acetonitrile and vacuum centrifuged to dryness.
- ABSC ammonium bicarbonate
- MALDI-MS analysis was conducted on a ABI DE-STR MALDI-TOF (laser settings set 1650-1850, 250 nsec delay). MS/MS of select mass-ions was performed on a 4700 Voyager MALDI-TOF-TOF(MS/MS laser setting:4000, CID gas off, 250 nsec delay) (Applied Biosystems). All analyses were conducted in reflectron mode. Samples were spotted on a stainless steel MALDI target using a "sandwich" spotting method wherein the analyte is spotted between two layers of alpha-cyano-4-hydroxycinnamic acid (Fluka). Assignments of the MS data to the nAChR sequence were performed using MASCOT® software.
- Figure 2 shows the MALDI-MS spectra for the peptide mass fingerprint of the delta (A), gamma (B), beta (C) and alpha (D) subunits of nAChR.
- the blue spectra represent the digests performed in the absence of BLEND 1
- the red spectra represent the digests performed in the presence of IX BLEND I.
- the sequence coverage without BLEND I is shown above the spectra
- the combined sequence coverage of the digests with and without BLEND I is shown above the respective nAChR subunit sequence.
- Figure 3 shows the sequences of the delta (A), gamma (B), beta (C) and alpha (D) subunits of nAChR. Amino acid sequences of transmembrane domains are underlined, and the amino acid residues of the peptide fragments identified by these experiments are shown in bold (SEQ ID NOS: 5-7).
- Opening up hydrophobic regions for MALDI-TOF MS is expected to reveal other pharmacologically relevant regions in areas that are normally unavailable for MALDI-TOF MS.
- Novel pharmacologically relevant regions may be found in previously inaccessible domains, including transmembrane and intramembranous domains.
- Labrou et al. J. Biol. Chem. 276:37944, 2001
- the Tachykinin NK2 receptor thus has sites where ligands bind, which are expected to be pharmacologically relevant, and these sites are in hydrophobic regions, which are the types of regions towards which the solubilizing agents of invention are directed.
- An additional MALDI-MS compatible surfactant that may be used as an alternative to or in combination with BLEND I is 250 mM NDSB-201 (a.k.a. Invitrogen MS- compatible solubilizer "B", Invitrosol B or 1MB).
- NDSB non-detergent sulfobetaine
- BSA bovine serum albumin
- MALDI-TOF-MS analysis was performed using an Applied Biosystems Voyager DE STR instrument.
- Samples 0.5 ⁇ L were spotted on the MALDI target using a "sandwich method" wherein 0.5 ⁇ L of alpha-cyano-4-hydroxycinnamic acid (Sigma- Aldrich, St.Louis, MO, USA) [7 mg/ml in 50% acetonitrile (Pierce)] with 0.1%TFA (Pierce) was first spotted on the plate and allowed to dry. An aliquot (0.5 ⁇ L) of sample was then spotted as an over- layer and allowed to dry.
- the PMF (peptide mass fingerprinting) of BSA was analyzed by Voyager Explorer software (Applied Biosystems, Foster City, CA, USA) as well as MASCOT® (Matrix Science, Boston, MA, USA).
- m/z 1083.6 two low abundance peaks (m/z 1083.6, 1283.7) are visible when 1MB is present (Spectrum B).
- the m/z 1083.6 signal corresponds to the peptide 161-168, which overlaps with 161-167 (m/z 927.5) as it is the result of a single missed cleavage site.
- the m/z 1283.7 (361-371) also overlaps with the corresponding sequence of an observed peptide peak (m/z 1439.8, 360-371).
- NDSB-201 may be used as an additive during "in-gel” proteolysis. Experiments with "in-gel” tryptic digests of Cytochrome P450 were carried out as described above except that NDSB-201 was present in the digest solution. As with BLEND I, 250 mM NDSB-201 enhanced sequence coverage by MALDI-MS over the control (44% for NDSB-201 versus 40% for the control).
- NDSB-201 prevents sodium from binding to BSA proteolysis products.
- An experiment was devised to investigate whether 250 mM NDSB-201 can titrate sodium adducts with increasing concentrations of NDSB-201 versus Na .
- the peptide Bradykinin (monoisotopic M. W. 998.5786) was used as a test peptide for the titration studies.
- Lys(-Des-Arg9, Leu8)-Bradykinin [H-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu-OH (C47H75N13O11) (SEQ ID NO:9)] from Calibrant II of InvitroMass LMW calibrant kit (Invitrogen) was diluted to a final 1 mM concentration in either deionized water (negative control) or in 250 mM NaCl (Sigma-Aldrich, St. Louis, MO). The calibrant was then mixed 1 :1 with various dilutions of NDSB-201 and spotted onto the MALDI target as described above. MALDI-TOF MS analysis was conducted in positive linear mode.
- Quantification of sodium sequestration by NDSB-201 was performed by analyzing the ion intensity of a centroid peak corresponding to the +Na adduct (m/z 1020.6) of the Bradykinin component (m/z 998.6). The average ion intensity of four spots was plotted against the respective concentration of 250 mM NDSB-201.
- FIG. 5 shows that, under these conditions, 100 mM NDSB-201 is sufficient to eliminate the formation of sodium and potassium adducts. Bradykinin was analyzed in the presence of 50 mM NaCl (A and B) or 50 mM KCl (C and D). The sodium adducts in (A) (m/z 1020.56, 1042.54), and the potassium adduct in (C) and (D) (M/z 1036.55), are identified by green font.
- the normalized intensity of the first sodium adduct peak (m/z 1020.56) was measured by the formula: (intensity of m/z 1020.56) / (intensity of m/z 998.58) + (intensity of m/z 1020.56), and plotted against the laser intensity (1500 - 1700).
- the values on the X axis represent laser intensity units.
- the resuts ( Figure 7) suggest that the relative intensities of unsodiated and sodiated Bradykinin are independent of the laser setting throughout the range tested.
- EXAMPLE 8 TESTING CHAOTROPIC ADDITIVES FOR USE WITH MS-
- the chaotropic mix be made fresh daily.
- the chaotrope concentration should not exceed about 0.35 M.
- solubilizer-MALDI samples are mixed with the MALDI matrix and applied to the MALDI target plate may require optimization.
- reasons for optimization may include the fact that surfactants are designed to break up intramolecular associations (thus preventing precipitation), whereas proper ionization by MALDI requires effective crystallization, a process that requires intramolecular contacts. Additionally, surfactants may lower the surface tension and inhibit droplet formation, and may thus dilute the sample across the surface area.
- a "sandwich" spotting protocol that may circumvent or limit these problems is as follows.
- a matrix solution is prepared, either a saturated solution of sinapinic acid in 50% acetonitrile/0.1% TFA for intact proteins, or a saturated solution of alpha-cyano-4- hydroxycinnamic acid in 50% acetonitrile/0.1% TFA, mixed 1 :1 with 50% acetonitrile/0.1% TFA for peptides.
- the matrix solution typically, 0.5 ⁇ L
- the matrix solution is spotted on the plate first. Once dry, the 0.5 ⁇ L of sample is spotted over it and allowed to dry. Then, another 0.5 ⁇ L of the matrix solution is spotted over that and dried.
- EXAMPLE 10 DEVELOPMENT OF SURFACTANT/DETERGENT BLENDS
- blends of NDSB-201 and detergents were tested, including those of the sulfobetaine class (such as SB 14 and SBlO) as well as other non-detergent sulfobetaines such as NDSB- 256.
- Each individual surfactant was tested by C 18-RP HPLC for its binding and elution properties. Components were evaluated based on their ability to (1) elute in the void volume or (2) elute in a distinct peak at a solvent concentration that is high enough to ensure separation from eluting peptides, and (3) their ability to not elute in subsequent runs as "ghost peaks".
- BLEND II is preferably used for liquid chromatography (LC), including without limitation HPLC and RP-HPLC.
- Blend II is also compatible with isoelectric focusing, including isoelectric separation methods that use immobilized pH gradients, such as immobilized pH gradient (IPG) strips, and pi-based separation methods such as capillary electrophoresis. Isoelectric focusing can also be performed using column chromatography . [00348] TABLE 8: FORMULATION OF Ix BLEND II
- Buffer exchanges using a Centricon device (Millipore) on Cytochrome P450 3A4 and 2D6 (Invitrogen/PanVera) were carried out in order to exchange the 20% glycerol included in the stock storage buffer for BLEND II.
- Cytochrome P450 was selected as a test subject because it contains a transmembrane segment within the first 30 N-terminal residues and thus requires a surfactant to be soluble in an aqueous solution.
- Buffer exchange was also carried out on affinity purified nicotinic acetylcholine receptor (nAChR) from Torpedo Pacifica electroplax organ reconstituted in mixed phospholipids vesicles in Tris buffer.
- nAChR nicotinic acetylcholine receptor
- sequence coverage is often the result of low solubility of peptides covering the hydrophobic regions of a protein. Even a soluble globular protein contains hydrophobic domains in its core.
- a sample containing 75 pmol of Cytochrome P450 1A2 was prepared and separated by SDS-PAGE using traditional protocols. A band at -60 kDa corresponding to P450 was excised and de-stained with 50% acetonitrile/ 25 mM ammonium bicarbonate pH 8.0. 200 ml of 100% acetonitrile was added to the gel piece and then dried down using a speed-vac apparatus. The sample was then rehydrated in a 10 ng/ml solution of trypsin (Promega) in 25 mM ammonium bicarbonate pH 8.0 plus Ix BLEND II and incubated overnight at 37 ⁇ C.
- trypsin Promega
- the digested peptides were extracted using one wash of 10 ml 5% FA, and an additional 10 ml 25% acetonitrile/5% FA wash.
- the extracted samples were analyzed by RP-HPLC and the resulting mass/ions identified by the Mascot sequence database search program (Matrix Science). Comparing the digested sample with and without BLEND II & LC/MS , we saw modest improvements in the sequence coverage (40% without and 48% with BLEND II).
- EXAMPLE 12 BLEND II : STABILITY STUDIES
- surfactant blends will be stable at room temperature for at least two weeks and three months at 2 0 C to 8 0 C. As initially provided, surfactant blends may be aliquoted and stored at -2O 0 C for eighteen months. Upon thawing, the blends may be diluted to reach the Ix concentration recommended for most purposes.
- EXAMPLE 13 BLEND II: REPRESENTATIVE KIT
- a representative BLEND II kit comprises a surfactant blend and a standard, separately contained and/or packaged, co-packaged in a box or other container.
- BLEND II is provided at 5x concentration in a clear polypropylene screw cap bottle. Sufficient BLEND II will be provided to perform ⁇ 75 detergent/buffer exchange procedures (5 ml).
- a representative standard is a Tryptic Digest of BSA standard prepared in BLEND II.
- a representative amount of standard is 25 ml of 1 ⁇ g/ ⁇ L BSA tryptic digest in BLEND II.
- the BLEND II solution and the standard are provided separately, each in a 1.7 ml polypropylene screw cap microcentrifuge vial with a removable cap (from, e.g., VWR).
- the kit may be warehoused at -2O 0 C and shipped at either -2O 0 C or 4 0 C. The customer will be advised to store the product at 4 0 C if it is to be used in under 2 months, or aliquoted at -2O 0 C until reaching the expiration date (18 months post production).
- the kit optionally includes a product manual which will cover storage conditions, protocols for several applications, and a link to an Invitrogen website that can keep the customer informed of new blends as well as new application protocols.
- EXAMPLE 14 BLEND II: PROTOCOLS
- BLEND II surfactant blends are formulated to be directly compatible with LC & LC/MS analysis at Ix concentration.
- the following protocol is designed for intact protein samples that contain solubilizers such as CHAPS, PEG, Glycerol, SDS, and salt concentration which may interfere with trypsin activity.
- the following protocol is intended to digest an intact protein with sequencing grade trypsin. This protocol is intended for proteins that have already undergone buffer exchange as described above. [00391] 1. Reconstitute lyophilized Trypsin using 25 mM Ammonium Bicarbonate at pH 8.0. Ideally, the final solution should have an enzyme to substrate ratio of 1 :25 -1 : 100 (w/w), and a final concentration of 25 mM ammonium bicarbonate.
- the sample may be analyzed directly by RP-HPLC using the following steps:
- One type of MS-compatible solubilizer is an alkyl glycoside having the structure
- Z can be O, S, Cl, I, Fl, Se, Br;
- MS-compatible solubilizer is a sulfobetaine having the structure
- R can be S, P or C; and [00422] x - 1-20.
- NDSBs Non-Detergent Sulfobetaines
- MS-compatible solubilizer is a non-detergent sulfobetaine having any of the following structures III- VI. [00424] [ I I I ]
- R is S, P or C.
- R is S, P or C.
- R is S, P or C.
- MS-compatible solubilizer is a bile acid having the structure
- R is a non-detergent sulfobetaine
- X can be H or OH.
- MS-compatible solubilizer is a Rabilloud detergent variant having the structure
- x 0-25, preferably 0-10;
- y 0-15, preferably 0-10;
- a representative kit comprising one or more of the MALDI-compatible solubilizers of the invention is packaged in a box and includes the following elements in a total of 2 solubilizing solutions in screw top bottles, 5 compositions in screw cap vials and, optionally, empty vials for sample manipulation. Deionized, sodium free water is used in the production of all solutions.
- A One (1) clear polypropylene screw cap bottle containing 10 ml of 5x BLEND I (formulation in Table 5) (sufficient detergent blend to perform -75 detergent/buffer exchange procedures);
- B One (1) clear polypropylene screw cap bottle containing 25 ml of 2x BLEND II (500 mM NDSB-201) (sufficient detergent blend to perform -75 detergent/buffer exchange procedures);
- C One (1) 1.7 ml polypropylene screw cap microcentrifuge vial (VWR International, West Chester, PA) containing 25 ⁇ L of 90 kDa InvitroMass standard (Invitrogen) (sufficient for at least 10 experiments); [00448] D. One (1) vial containing 20 mg of sinapinic acid; [00449] E. One (1) vial containing 20 mg of alpha-cyano-4-hydroxycinnamic acid; [00450] F. One (1) 1.7 ml polypropylene screw cap microcentrifuge vial with red cap (VWR) containing 1 ml of 0.1% trifluoroacetic acid (solvent for mixing MALDI matrices); [00451] G.
- Invitrosol-MALDI surfactant blends are formulated to be directly compatible with MALDI-MS analysis at IX concentration.
- the following protocol is designed for intact protein samples that do not contain any interfering contaminants such as CHAPS, PEG or SDS. Please refer to the "Buffer Exchange Protocol” below if your sample contains any of these materials.
- Invitrosol-MALDI blends can be exchanged for commonly used surfactants that are incompatible with MS.
- the following exemplary protocol is designed to remove buffer solution components that may interfere with MALDI-MS analysis.
- the sample may be analyzed directly by MALDI-MS using the protocol described above in steps 2 through 7 for "Direct Analysis and Calibration of Intact Hydrophobic Proteins".
- the following protocol is intended to digest an intact protein with sequencing grade trypsin. This protocol is intended for proteins that have already undergone buffer exchange as described above. [00478] 1. Mix your intact protein sample from the "Buffer Exchange Protocol" (above) with ammonium bicarbonate pH 8 and trypsin solution. Ideally the final solution should have an enzyme to substrate ratio of 1 :25 -1 :250, and a final concentration of 50 raM ammonium bicarbonate.
- EXAMPLE 18 MAXION AC: EFFECT OF SILICA ADDITIVE ON SIGNAL-
- a resin solution (20 mg/ml) was prepared by dissolving 20 mg of Lichrosorb Si60 (5 ⁇ m) beads (EMD) in 1 ml of a solution of resin diluent [99% (v /v) HPLC grade acetonitrile and 0.01% (v/v) ACS grade ammonium hydroxide].
- the matrix and resin solutions were mixed 9: 1 (v:v) to generate a matrix/resin solution.
- two (2) ⁇ L of conventional matrix solution or matrix/resin solution were combined with 1 ⁇ L of sample before spotting.
- Figure 14 shows the comparative analysis by MALDI-MS.
- the top two spectra show MALDI-MS analyses of a calibrant mixture [Bradykinin fragments (1-5, m/z 573.67; 1- 7, m/z 757.87), Bradykinin Lys (-Des-Arg9, Leu8) (m/z 998.58), ACTH (1-16, m/z 1936.99), ACTH (1-24, m/z 2932.59)] in conventional alpha-cyanno (top left spectrum) and the alpha- cyano:silica mixture (top right spectrum).
- the total ion counts on the y axes indicate the improved signal to noise obtained with silica resin. There are significant gains in signal-to- noise across the spectrum despite the total ion counts decreasing from 2.3e4 to 1.7e4 at the same laser intensity.
- Maxlon AC silica resin yielded higher sequence coverage than conventional CHCA.
- Maxlon AC silica resin also yielded higher MASCOT scores for all proteins except ovalbumin, although Maxlon AC silica resin did yield higher sequence coverage.
- the reason is that the ovalbumin sample is not homogeneous (Sigma-Aldrich) and Maxlon "amplifies" the peak intensity of multiple lower-abundance ovalbumin isoforms ( Figure 15; isoforms are color coded to mass ions).
- the sequence coverage for ovalbumin is higher, the increased number of "contaminant peaks" resulted in a lower confidence score by MASCOT, which searches for single accession numbers per spectrum.
- the lower spectrum has a number of peaks corresponding to beta- galactosidase peptides that are suppressed by the high salt concentration in the top spectrum.
- the peaks from both spectra were analyzed by the database search application MASCOT (Matrix Science) and Maxlon AC silica resin yielded a score of 147 and sequence coverage of 37%.
- MASCOT scores >57 are statistically significant.
- Maxlon AC silica resin can deliver greater signal-to-noise, which results in better sensitivity and protein identifications with improved confidence and sequence coverage.
- the non-volatile matrix additive can be silica or a compound containing silica.
- Such compounds include without limitation silicon dioxide (SiO 2 ), silicon carbide, (SiC) and silicates.
- silicon refers to a tetravalent nonmetallic element (Si) that occurs combined as the most abundant element next to oxygen in the earth's crust. Natural silicon dioxide (SiO 2 ) occurs in crystalline, amorphous and impure forms (quartz, opal and sand respectively).
- LiChrospher® is an irregular porous packing material manufactured in Germany by E. Merck.
- LiChrosorb® comprises porous irregular silica particles are finely classified in the 5 ⁇ m, 7 ⁇ m and 10 ⁇ m range.
- LiChrosorb® is available with different modifications, such as polar derivatives (Si60 and SiIOO), non-polar derivatives (RP-8, RP- 18 and RP-select B), and derivatives of medium polarity (NH 2 , CN and DIOL).
- LiChroprep® comprises porous irregular silica particles are finely classified in the 15-25 ⁇ m, 25-40 ⁇ m and 40-63 ⁇ m range.
- LiChrospher® (the 40 ⁇ m material is a.k.a. Superspher® in Europe) comprises particles that are more regular than LiChrosorb.
- LiChrospher® is available with different modifications, such as the polar modified derivatives LiChrospher® CN, LiChrospher® NH 2 and LiChrospher® DIOL, as well as LiChrospher® Si.
- Purospher® RP-18 is based upon a high purity, metal free silica. It is relatively chemically stable.
- Silicates are arrangements of the elements silicon and oxygen with a wide variety of other elements (most common silicates are quartz and feldspars).
- the invention can be practiced with silicates other than silica if they are prepared in the proper form and/or ground to an appropriate particle size (see below).
- a MS-compatible sorbent of the invention can be silica; alumina; titanium; tin; germanium oxide; an indium tin oxide; a metal oxide; a chloride; a sulfate; a phosphate; a carbonate; a fluoride; a polymer-based oxide, chloride, sulfate, carbonate, phosphate or fluoride; diatomaceous earth; graphite or activated charcoal; gold; or activated gold.
- alumina refers to various forms of aluminum oxide, including the naturally occurring corundum.
- Tin, titanium and alumina have been examined (data not shown) and, to some degree, all have the desirable characteristics of silica as regards MS-MALDI analysys, i.e., they reduce noise, reduce or eliminate adduction, provide for a greater density and more even distribution of crystals and co-crystals on a MALDI target plate, and the like.
- the MS-compatible sorbents of the invention can be inorganic sorbents.
- Sorbents are insoluble, or partially or practically insoluble, materials or mixtures of materials used to recover liquids through the mechanism of absorption, or adsorption, or both.
- partially insoluble it is meant that the sorbent is at least 50% or about 50% insoluble in excess fluid, preferably 70% or about 70%, most preferably 80% or about 80% insoluble.
- a practically insoluble substance is at least about 85%, preferably 90% or about 90%, most preferably 95% or about 95% insoluble in excess fluid.
- An insoluble substance is at least about 98%, preferably 99% or about 99%, most preferably 100% or about 100% insoluble in excess fluid.
- Absorbents are materials that pick up and retain liquid distributed throughout their molecular structure causing the material to swell as much as 50% or about 50%, or more.
- synthetic sorbents may be used. These include polymer-based sorbents, such as Tenax-GC and Tenax-TA, which are available in various mesh sizes (20-35, 35-60, 60-80, and 80-100 microns, for example) from Scientific Instrument Services, Inc. (Ringoes, NJ).
- the Tenax compositions comprise poly(2,6-diphenyl- 1 ,4-phenylene oxide).
- the matrix additive can be provided in a variety of forms, typically as a colloid, such as a colloidal suspension, a resin or slurry.
- the additive can be in the form of at least partially suspended beads.
- the composition and particle size of any given non- volatile matrix additive will influence the choice of colloid. Particle size influences other factors as well. For example, powdered (fumed) silica disrupts crystal and co-crystal formation, presumably due to the extremely small particle size ( ⁇ 1 micron).
- the concept of particle size encompasses several characteristics, including without limitation particle shape, mean particle size and particle size distribution.
- the particles can be spherical beads as well as more irregular particles.
- DlO is the size of a pore through which 10% of the particles pass through (10% of the particles are smaller than the pore size); D50 is the point at which 50% of the particles are smaller; D90 is the point at which 90% of the particles are smaller; and so on.
- gradation index is used to indicate the degree of uniformity for the distribution of particle sizes.
- the ratio of D90/D10 is used to evaluate GI herein, but other ratios can be established for any given field or application.
- the GI (D90/D10) is ⁇ 10 or about 10, preferably the GI ⁇ 5 or about 5, more preferably the GI ⁇ _3 about 3, and most preferably, the GI ⁇ 2.5 or about 2.5, or ⁇ 2 or about 2.
- Table 11 lists some forms of silica that meet such criteria and thus may be used to practice the invention.
- the LiChrosorb® compositions are commercially available from EMD Biochemicals/Calbiochem (San Diego, CA). TABLE 11 : PARTICULATE FORMS OF SILICA
- MOPS 3-(N-Morpholino)propanesulfonic acid
- FIG. 20 illustrates an example of a MALDI-TOF analysis of a high molecular weight protein and how Maxlon SA can enhance the signal intensities of low abundance proteins.
- Spectrum A shows the prevalence of noise and the resulting perturbation of the baseline.
- Spectrum B shows how Maxlon SA can practically eliminate the baseline perturbation and markedly improve the signal-to-noise of the analyte peaks. Further, Maxlon SA allows the improved mass measurement of the dimer peak, and even the identification of the trimer (insets).
- Figure 21 shows the chemical structures of the MES (2-(N-)
- Morpholino)ethanesulfonic Acid MOPS (3-(N-Morpholino)propanesulfonic Acid), MOPSO (3-(N-Morpholino)-2-hydroxypropanesulfonic Acid) and MOBS (4-(N- Morpholino)butanesulfonic Acid) buffers. It is believed that these and other morpholino- sulfonic acids, as well as other related compounds, may also be useful as MALDI matrix additives.
- One type of matrix additive of the invention has the structure [00514] [ IX]
- the non-volatile MALDI matrix additives are comprised within a solution into which the MALDI matrix is dissolved or diluted.
- a Maxlon SA Matrix Diluent comprises, by way of non-limiting example,50% acetonitrile (v/v) (HPLC grade); 0.1% TFA (v/v) (HPLC grade); and 40 mM MES, in a final volume of 1.1 ml.
- An exemplary Maxlon AC Matrix Diluent comprises, by way of non-limiting example, 80% acetonitrile (v/v) (HPLC grade) and 0.05% TFA (v/v) (HPLC grade), in a final volume of 1.1 ml.
- An exemplary 1OX stock solution of Maxlon AC Resin comprises 20 mg of Lichrosorb Si60 (5 ⁇ m beads) silica.
- the Maxlon AC Resin Diluent comprises 99% acetonitrile (v/v) and 0.01% ammonium hydroxide (v/v) (ACS grade) in a final volume of 1.1 ml.
- the CHCA matrix may be provided as, by way of non-limiting example, 2.2 mg of CHCA in a 1.5 ml eppendorf tube. This can be prepared by adding 100 ⁇ L of a solution of 22 mg/ml of CHCA in 100% methanol (HPLC grade) to a 1.5 ml eppendorf tube, and then drying to remove the menthol, e.g., by using a speed-vac with no heat.
- the Maxlon AC Cation Sequestration Diluent (5X) is 500 mM NDSB in a final volume of 1.1 ml.
- Calbiochem® is a brand of EMD Biosciences, Inc. (San Diego, CA).
- kits of the invention comprises Maxlon SA and/or Maxlon AC.
- kit components include without limitation:
- MALDI matrices e.g., SA and CHCA
- kits are contemplated by this aspect of the invention: Maxlon SA, Maxlon AC and Maxlon Complete.
- An exemplary Maxlon SA kit geared towards applications wherein SA is the MALDI matrix, contains five 1.5 ml eppendorff vials of Sinapinic Acid (20 mg), five 1.5 ml eppendorff vials of SA diluent (40 mM MES, 50% acetonitrile, 0.1% TFA), and one 0.5 ml eppendorff vial with InvitroMass LMW Calibrant mix 4 (Invitrogen).
- An exemplary Maxlon AC kit geared towards applications wherein CHCA is the MALDI matrix, contains five 1.5 ml eppendorff vials of CHCA (2.2 mg), five 1.5 ml eppendorff vials of CHCA diluent 1 (80% acetonitrile, 0.1% TFA), one 1.5 ml eppendorff vial of Maxlon AC Resin (20 mg of silica beads in 99% acetonitrile, 0.01% ammonium hydroxide), one 1.5 ml of Cation Sequestration Diluent (1 ml of 500 mM NDSB), and one 0.5 ml eppendorff tube of InvitroMass LMW Calibrant mix 2 (Invitrogen).
- a Maxlon Complete kit which can be used with a variety of MALDI matrices, contains the contents of the Maxlon SA and Maxlon AC kits.
- Different calibrants can alternatively or additionally be included. These include without limitation the InvitromassTM calibrants from Invitrogen.
- the InvitromassTM Calibrant 1 set (500-1000 Da) comprises Bradykinin fragment (aa 1-5), Bradykinin fragment (aa 1-7) and Lys(-Des-Arg9, Leu8)-Bradykinin.
- the InvitromassTM Calibrant 2 set (1000-3000 Da) comprises Lys(-Des-Arg9, Leu8)-Bradykinin; ACTH fragment (aa 1-16); and ACTH fragment (aa 1-24).
- the InvitromassTM Calibrant 3 set (3000-6000 Da) comprises ACTH fragment (aa 1-24); ACTH fragment (aa 1-39); and Insulin.
- the InvitromassTM Calibrant 4 set (6000-12000 Da) comprises Insulin, Ubiquitin and Cytochrome C.
- Maxlon AC silica demonstrated a diminished ability to overcome the suppressive effects of sample salts.
- the low pH in the matrix solution was promoting protonation of the SiO moieties.
- the Maxlon AC loses potency after 24 hrs in an acidic solution (0.05%TFA ⁇ pH 2.5).
- the resin and CHCA are mixed immediately prior to use, and this solution is used within about 24, about 36, or preferably, 48 hours.
- Figure 22 shows MALDI-MS spectra of InvitroMass LMW Calibrant 2 (diluted 1 :200 in 100 mM NaCl) in CHCA and CHCA mixed with silica under different storage conditions.
- the results suggest that even after 8 days at 37 0 C, the silica resin performs as well as freshly prepared resin.
- the Resin Diluent formulation should still include 0.01% AmOH to protect the resin from unforeseen changes in pH during storage conditions. Based on the stability studies described above, we extrapolate that the resin slurry is stable up to 8 months when stored at -20°C.
- Maxion SA Diluent is 50% acetornitrile (HPLC grade) (v/v); 0.1% (v/v) TFA (HPLC grade); and 40 mM MES, in a final volume of 1.1 ml).
- HPLC grade acetonitrile HPLC Grade >99.93% acetonitrile (Sigma-Aldrich Corp., St. Louis, MO, catalog No. 270717).
- HPLC grade TFA is Trifluoroacetic Acid, Sequanal Grade (Pierce Biotechnology, Rockford, IL, catalog No. 28904).
- MES 2-(N-Morpholino)ethanesulfonic acid, monohydrate (Research Organics, Cleveland, OH, catalog No. 0113M).
- Edge bias has been reduced or eliminated, and nearly the entire surface (as opposed to patches of the surface) of the plate consists of "sweet spots”.
- the following experiment was carried out using the MALDI plates shown in Figure 23. Rather than selecting and focusing "sweet spots" during acquisition and scanning of the plate lacking silica (A in Figure 23), the laser's target point was moved about randomly on both plates. The resultant spectra ( Figure 24) show that, under these conditions, only the targets prepared with silica yield useful spectra. Indeed, the spectrum of the sample without silica is not even sufficient to result in identification of the protein analyte in the absence of targeting to sweet spots on the target plate.
- EXAMPLE 30 PEPTIDE IDENTIFICATION USING AN MS-COMPATIBLE
- EPG immobilized pH gradients
- Cytochrome P450 and nAChR were digested with trypsin (Promega) or endoproteinase AspN (Roche) overnight in the presence of an MS-compatible solubilizer blend of 25 mM NDSB-201, 25 mM NDSB-256, 0.22 mM SB-14, 25 mM Ammonium bicarbonate, pH 7.8. 5 ug of the digested samples were loaded directly on 3-10 NL IPG strips (Invitrogen) in addition to a 1 ug aliquot of myoglobin. After electrophoretic focusing, the strips were cut into 8 equal pieces and peptides were extracted using 5% TFA and 50%ACN/2.5% TFA.
- the extracted peptides were dried using speed vac (Savant) and were reconstituted in 20% ACN/2.5% FA.
- the samples were then analyzed with both 4700 MALDI-TOF/TOF (Applied Biosystems) and Q-TOF LC-ESI/MS (Waters).
- the data were analyzed by GPS explorer (Applied Biosystems), Mascot Distiller (Matrix Sciences) sequence database search and GPMAW 6.0 (ChemSW).
- Sequence assignments were made based on 1) the exact mass of the peptide, and 2) by comparing agreement between the predicted pi and the known pi of a myoglobin proteolytic fragment co-migrating within the same pi bin.
- Residues 299-311 (M2-M3 loop) LPETALAVPL IG
- Residues 180-203 (ligand binding domain) VSISPESDRP DLSTFMESGE WVMK
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Cited By (4)
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WO2012028525A3 (fr) * | 2010-08-30 | 2012-10-26 | F. Hoffmann-La Roche Ag | Procédé de production d'une particule lipidique, particule lipidique elle-même et son utilisation |
US8449744B2 (en) | 2007-11-12 | 2013-05-28 | Becton, Dickinson And Company | MS-compatible nonionic or zwitterionic surfactants in free-flow electrophoresis |
US8791063B2 (en) | 2011-08-25 | 2014-07-29 | Hoffmann-La Roche, Inc. | Shortened tetranectin-apolipoprotein A-I fusion protein, a lipid particle containing it, and uses thereof |
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WO2009023158A2 (fr) * | 2007-08-10 | 2009-02-19 | University Of Massachusetts | Micelles inverses polymères comme agents d'extraction sélectifs et procédés associés d'analyse en sm maldi |
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JP7070671B2 (ja) * | 2018-04-16 | 2022-05-18 | 株式会社島津製作所 | 質量分析用キット、微生物識別用キット、試料の調製方法、分析方法および微生物の識別方法 |
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CA2243230A1 (fr) * | 1998-07-15 | 2000-01-15 | Aled Edwards | Dispositif et methode pour la determination des limites du domaine des proteines |
AU2001234880A1 (en) * | 2000-02-08 | 2001-08-20 | The Regents Of The University Of Michigan | Mapping of differential display of proteins |
US7700306B2 (en) * | 2002-04-04 | 2010-04-20 | Electrophoretics Limited | Method for charcterising analytes |
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2009
- 2009-09-02 US US12/553,017 patent/US20100187475A1/en not_active Abandoned
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US20030036097A1 (en) * | 2001-08-17 | 2003-02-20 | Aftab Alam | Urea containing reagents for treatment of proteins |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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US8449744B2 (en) | 2007-11-12 | 2013-05-28 | Becton, Dickinson And Company | MS-compatible nonionic or zwitterionic surfactants in free-flow electrophoresis |
WO2012028525A3 (fr) * | 2010-08-30 | 2012-10-26 | F. Hoffmann-La Roche Ag | Procédé de production d'une particule lipidique, particule lipidique elle-même et son utilisation |
JP2013538205A (ja) * | 2010-08-30 | 2013-10-10 | エフ.ホフマン−ラ ロシュ アーゲー | テトラネクチン−アポリポタンパク質a−i粒子を産生するための方法、それを用いて得られる脂質粒子、及びその使用 |
US8791063B2 (en) | 2011-08-25 | 2014-07-29 | Hoffmann-La Roche, Inc. | Shortened tetranectin-apolipoprotein A-I fusion protein, a lipid particle containing it, and uses thereof |
US9139640B2 (en) | 2011-08-25 | 2015-09-22 | Hoffmann-La Roche Inc. | Shortened tetranectin-apolipoprotein A-1 fusion protein, a lipid particle containing it, and uses thereof |
CN113866325A (zh) * | 2021-09-14 | 2021-12-31 | 马鞍山市产品质量监督检验所 | 一种绿色环保型测定饮料中多种添加剂的分析方法 |
CN113866325B (zh) * | 2021-09-14 | 2023-05-05 | 马鞍山市产品质量监督检验所 | 一种测定饮料中多种添加剂的分析方法 |
Also Published As
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WO2006047614A3 (fr) | 2007-05-31 |
US20100187475A1 (en) | 2010-07-29 |
US20110315930A1 (en) | 2011-12-29 |
US20060214104A1 (en) | 2006-09-28 |
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